Methods and compositions for consumables

ABSTRACT

Provided herein are methods and compositions related to plant based meat substitutes which have properties similar to meat.

PRIORITY CLAIM

This application is a continuation of U.S. application Ser. No.14/152,499 filed Jan. 10, 2014, which is a continuation ofPCT/US2012/046560 filed Jul. 12, 2012, which claims priority to U.S.application Ser. No. 61/572,205 filed Jul. 12, 2011, all of which areincorporated herein in their entirety by reference.

BACKGROUND OF THE INVENTION

Animal farming has a profound negative environmental impact. Currentlyit is estimated that 30% of Earth's land surface is dedicated to animalfarming and that livestock account for 20% of total terrestrial animalbiomass. Due to this massive scale animal farming accounts for more than18% of net greenhouse gas emissions. Animal farming may be the largesthuman source of water pollution, and animal farming is by far theworld's largest threat to biodiversity. It has been estimated that ifthe worlds human population could shift from a meat containing diet to adiet free of animal products, 26% of Earth's land surface would be freedfor other uses. Furthermore the shift to a vegetarian diet wouldmassively reduce water and energy consumption.

The consumption of meat has a profound negative impact on human health.The heath benefits of a vegetarian diet are well established. If thehuman population would shift to a vegetarian diet the cost savings inhealth care would be significant.

Hunger is a worldwide problem, yet the world's 4 major commodity crops(soybeans, maize, wheat, and rice) already supply more than 100% of thehuman population's requirements for calories and protein, includingevery essential amino acid.

Plant based meat substitutes have largely failed to cause a shift to avegetarian diet. The current state of the art for meat substitutecompositions involves the extrusion of soy/grain mixture, resulting inproducts which largely fail to replicate the experience of cooking andeating meat. Common limitations of these products are a texture andmouthfeel that are more homogenous than that of equivalent meatproducts. Furthermore, as the products must largely be sold pre-cooked,with artificial flavors and aromas built in, they fail to replicatearomas, flavors, and other key features associated with cooking meat. Asa result, these products appeal mainly to a limited consumer base thatis already committed to vegetarianism/veganism, but have failed toappeal to the larger consumer segment accustomed to eating meat.

Disclosed herein are improved methods and compositions which moreaccurately replicate the characteristics that consumers value in thepreparation and consumption of meat and which overcome the shortcomingsand drawbacks of current meat substitutes.

SUMMARY OF THE INVENTION

In some aspects, the invention provides a meat substitute compositioncomprising a protein content, wherein one or more isolated and purifiedproteins accounts for 10% or more of said protein content by weight,wherein said meat substitute composition accurately mimics the taste,texture, or color of a meat product derived from animal sources.

In one embodiment, the meat substitute composition accurately mimics thecolor of said meat product in its raw state and in a cooked state aftercooking.

In another embodiment, the one or more isolated and purified proteinsaccounts for 25% or more of said protein content by weight.

In another embodiment, the one or more isolated and purified proteinsaccounts for 50% or more of said protein content by weight.

In another embodiment, the one or more isolated and purified proteinsaccounts for 75% or more of said protein content by weight.

In another embodiment, the one or more isolated and purified proteinsaccounts for 90% or more of said protein content by weight.

In another embodiment, gluten does not account for 10% or more of saidprotein content by weight.

In another embodiment, each of said isolated, purified proteins isseparately isolated and purified.

In another embodiment, the meat substitute composition comprises 1-7isolated and purified proteins.

In another embodiment, said 1-7 isolated and purified proteins are eachisolated from different plant species.

In some embodiments the meat substitute comprises less than 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 individual proteinsisolated from one or more plant species.

In another embodiment, said protein content comprises no more than traceamounts of any other proteins derived from the one or more plantspecies.

In another embodiment, said one or more isolated and purified proteinsare selected from the group consisting of leghemoglobin, non-symbiotichemoglobin, hemoglobin, myoglobin, chlorocruorin, erythrocruorin,neuroglobin, cytoglobin, protoglobin, truncated 2/2 globin, HbN,cyanoglobin, HbO, Glb3, and cytochromes, Hell's gate globin I, bacterialhemoglobins, ciliate myoglobins, flavohemoglobins, ribosomal proteins,actin, hexokinase, lactate dehydrogenase, fructose bisphosphatealdolase, phosphofructokinases, triose phosphate isomerases,phosphoglycerate kinases, phosphoglycerate mutases, enolases, pyruvatekinases, glyceraldehyde-3-phosphate dehydrogenases, pyruvatedecarboxylases, actins, translation elongation factors,ribulose-1,5-bisphosphate carboxylase oxygenase (rubisco),ribulose-1,5-bisphosphate carboxylase oxygenase activase (rubiscoactivase), albumins, glycinins, conglycinins, globulins, vicilins,conalbumin, gliadin, glutelin, gluten, glutenin, hordein, prolamin,phaseolin (protein), proteinoplast, secalin, extensins, triticeaegluten, zein, any seed storage protein, oleosins, caloleosins,steroleosins or other oil body proteins, vegetative storage protein A,vegetative storage protein B, moong seed storage 8S globulin.

In another embodiment, said one or more isolated and purified proteinsare not isolated from an animal.

In another embodiment, said one or more isolated and purified proteinsare isolated from a single plant source.

In another embodiment, said one or more isolated and purified proteinsare isolated from multiple plant sources.

In another embodiment, wherein said one or more isolated, purifiedproteins are isolated from a genetically modified organism.

In some embodiments, said genetically modified organism is a geneticallymodified bacteria or yeast organism.

In some embodiments, said isolated, purified protein has been formedinto fibers.

In particular embodiments, said fibers resemble skeletal muscle fibers.

In yet more particular embodiments, said fibers are asymmetric fibers.

In some embodiments, the meat substitute composition further comprisesone or more isolated and purified iron-containing proteins.

In some embodiments, said one or more isolated and purifiediron-containing proteins is selected from the group consisting ofhemoglobin, myoglobin, leghemoglobin, non-symbiotic hemoglobin,chlorocruorin, erythrocruorin, neuroglobin, cytoglobin, protoglobin,truncated 2/2 globin, HbN, cyanoglobin, HbO, Glb3, and Hell's gateglobin I, bacterial hemoglobins, ciliate myoglobins, flavohemoglobins.

In a particular embodiment, said iron-containing protein comprises anamino acid sequence with at least 70% homology to SEQ ID NO 1. SEQ ID NO1: MVAFTEKQDALVSSSFEAFKANIPQYSVVFYTSILEKAPAAKDLFSFLANGVDPTNPKLTGHAEKLFALVRDSAGQLKASGTVVADAALGSVHAQKAVTDPQFVVVKEALLKTIKAAVGDKWSDELSRAWEVAYDELAAAIKKA.

In a particular embodiment, said iron-containing protein comprises anamino acid sequence with at least 70% homology to SEQ ID NO 2. SEQ ID NO2: MIDQKEKELI KESWKRIEPN KNEIGLLFYA NLFKEEPTVS VLFQNPISSQ SRKLMQVLGILVQGIDNLEG LIPTLQDLGR RHKQYGVVDS HYPLVGDCLL KSIQEYLGQG FTEEAKAAWTKVYGIAAQVM TAE. In some embodiments said iron-containing proteincomprises an amino acid sequence with at least 80% homology to SEQ ID NO2. In some embodiments said iron-containing protein comprises an aminoacid sequence with at least 90% homology to SEQ ID NO 2. In someembodiments said iron-containing protein comprises an amino acidsequence with at least 98% homology to SEQ ID NO 2.

In a particular embodiment, said iron-containing protein comprises anamino acid sequence with at least 70% homology to SEQ ID NO 3. SEQ ID NO3: MRKQPTVFEK LGGQAAMHAA VPLFYKKVLA DDRVKHYFKN TNMEHQAKQQ EDFLTMLLGGPNHYKGKNMA EAHKGMNLQN SHFDAIIENL AATLKELGVS DQIIGEAAKV IEHTRKDCLG K. Insome embodiments said iron-containing protein comprises an amino acidsequence with at least 80% homology to SEQ ID NO 3. In some embodimentssaid iron-containing protein comprises an amino acid sequence with atleast 90% homology to SEQ ID NO 3. In some embodiments saidiron-containing protein comprises an amino acid sequence with at least98% homology to SEQ ID NO 3.

In some embodiments, the isolated and purified proteins are assembledinto one or more gels.

In some embodiments, the meat substitute composition further comprisesone or more fats.

In particular embodiments, said one or more fats are derived from aplant source.

In another aspect, the invention provides a meat substitute product thatcomprises an indicator that indicates cooking progression from a rawstate to a cooked state, wherein said meat substitute product is derivedfrom non-animal sources.

In some embodiments, said indicator is a visual indicator thataccurately mimics the color transition of a meat product during saidcooking progression.

In one embodiment, said color transition is from red to brown.

In one embodiment, said color transition is from pink to white or tan.

In one embodiment, said visual indicator transitions from a translucentto opaque color during said cooking progression.

In some embodiments, the indicator is an olfactory indicator thatindicates cooking progression.

In one embodiment, said olfactory indicator is one or more volatileodorants released during cooking.

In some embodiments, said indicator comprises one or more isolated,purified iron-containing proteins.

In particular embodiments, said one or more isolated, purifiediron-containing proteins is in a reduced state before cooking.

In one embodiment, said one or more isolated and purifiediron-containing proteins is selected from the group consisting ofhemoglobin, myoglobin, leghemoglobin, non-symbiotic hemoglobin,chlorocruorin, erythrocruorin, neuroglobin, cytoglobin, protoglobin,truncated 2/2 globin, HbN, cyanoglobin, HbO, Glb3, and cytochromes,Hell's gate globin I, bacterial hemoglobins, ciliate myoglobins,flavohemoglobins.

In a particular embodiment, said iron-containing protein comprises anamino acid sequence with at least 70% homology to SEQ ID NO 1. SEQ ID NO1: MVAFTEKQDALVSSSFEAFKANIPQYSVVFYTSILEKAPAAKDLFSFLANGVDPTNPKLTGHAEKLFALVRDSAGQLKASGTVVADAALGSVHAQKAVTDPQFVVVKEALLKTIKAAVGDKWSDELSRAWEVAYDELAAAIKKA.

In some embodiments, said one or more isolated and purifiediron-containing proteins are not isolated from an animal. In someembodiments compositions of the invention do not contain any proteinsfrom an animal.

In particular embodiments, said one or more isolated and purifiediron-containing proteins are isolated from one or more plant sources.

In yet more particular embodiments, said one or more isolated, purifiedproteins are isolated from the root nodules, roots, seeds, leaves, orstems of said one or more plant sources.

In other particular embodiments, said one or more plant sources are soyor pea plants.

In one embodiment, said one or more plant sources comprises one or moreplants of the legume family.

In some embodiments, said one or more isolated and purified ironcarrying proteins in a reduced or oxidized state has a similar UV-VISprofile to a myoglobin protein derived from an animal source when in anequivalent reduced or oxidized state.

In a particular embodiment, the difference between the peak absorbancewavelength of said one or more isolated and purified iron-containingproteins and the peak absorbance wavelength of myoglobin derived from ananimal source is less than 5%.

In some embodiments, said one or more isolated, purified proteins areisolated from a genetically modified organism.

In one embodiment, said genetically modified organism is a geneticallymodified bacteria or yeast organism.

In some embodiments, the meat substitute product contains nomethylcellulose, no carrageenan, no caramel color, no konjac flour, nogum arabic, and no acacia gum.

In particular embodiments, the meat substitute product additionallycontains less than 1% wheat gluten.

In a more particular embodiment, said meat substitute product containsno wheat gluten.

In other particular embodiments, said meat substitute product containsno soy protein isolate.

In other particular embodiments, said meat substitute product containsno soy protein concentrate.

In other particular embodiments, said meat substitute product containsno soy protein.

In a more particular embodiment, said meat substitute product containsless than 5% carbohydrates.

In other particular embodiments, said meat substitute product containsno tofu.

In some embodiments, said meat substitute product contains no tofu, andno wheat gluten.

In some embodiments, said meat substitute product contains no soyprotein, and no wheat gluten.

In some embodiments, said meat substitute product contains no animalproducts and less than 5% carbohydrates.

In some embodiments, said meat substitute product contains less than 1%cellulose.

In some embodiments, said meat substitute product contains less than 5%insoluble carbohydrates.

In some embodiments, said meat substitute product contains no soyprotein, and less than 1% cellulose.

In some embodiments, said meat substitute product contains no soyprotein, and less than 5% insoluble carbohydrates.

In some embodiments, said meat substitute product contains no wheatgluten, and less than 1% cellulose.

In some embodiments, said meat substitute product contains no wheatgluten, and less than 5% insoluble carbohydrates.

In another aspect, the invention provides a muscle tissue replicacomprising a protein content, wherein said protein content comprises oneor more isolated and purified proteins, wherein said muscle tissuereplica approximates the taste, texture, or color of an equivalentmuscle tissue derived from an animal source.

In some embodiments, said one or more isolated and purified proteinsaccounts for at least 50% of said protein content by weight. In someembodiments, said one or more isolated and purified proteins accountsfor at least 40% of said protein content by weight. In some embodiments,said one or more isolated and purified proteins accounts for at least30% of said protein content by weight. In some embodiments, said one ormore isolated and purified proteins accounts for at least 20% of saidprotein content by weight. In some embodiments, said one or moreisolated and purified proteins accounts for at least 10% of said proteincontent by weight.

In some embodiments, said one or more isolated and purified proteinsaccounts for at least 50% of said composition content by weight. In someembodiments, said one or more isolated and purified proteins accountsfor at least 40% of said composition content by weight. In someembodiments, said one or more isolated and purified proteins accountsfor at least 30% of said composition content by weight. In someembodiments, said one or more isolated and purified proteins accountsfor at least 20% of said composition content by weight. In someembodiments, said one or more isolated and purified proteins accountsfor at least 10% of said composition content by weight. In someembodiments, said one or more isolated and purified proteins accountsfor at least 5% of said composition content by weight. In someembodiments, said one or more isolated and purified proteins accountsfor at least 1% of said composition content by weight.

In some embodiments, said protein content is derived from one or morenon-animal sources.

In particular embodiments, said one or more non-animal sources is aplant source.

In other particular embodiments, said one or more non-animal sources isa genetically modified yeast or bacteria.

In some embodiments, each of said one or more isolated proteins isisolated and purified separately.

In some embodiments, said one or more isolated proteins are selectedfrom the group consisting of hemoglobin, myoglobin, chlorocruorin,erythrocruorin, neuroglobin, cytoglobin, protoglobin, truncated 2/2globin, HbN, cyanoglobin, HbO, Glb3, and cytochromes, Hell's gate globinI, bacterial hemoglobins, ciliate myoglobins, flavohemoglobins,ribosomal proteins, actin, hexokinase, lactate dehydrogenase, fructosebisphosphate aldolase, phosphofructokinases, triose phosphateisomerases, phosphoglycerate kinases, phosphoglycerate mutases,enolases, pyruvate kinases, glyceraldehyde-3-phosphate dehydrogenases,pyruvate decarboxylases, actins, translation elongation factors,ribulose-1,5-bisphosphate carboxylase oxygenase (rubisco),ribulose-1,5-bisphosphate carboxylase oxygenase activase (rubiscoactivase), albumins, glycinins, conglycinins, globulins, vicilins,conalbumin, gliadin, glutelin, gluten, glutenin, hordein, prolamin,phaseolin (protein), proteinoplast, secalin, extensins, triticeaegluten, zein, any seed storage protein, oleosins, caloleosins,steroleosins or other oil body proteins, vegetative storage protein A,vegetative storage protein B, moong seed storage 8S globulin.

In one embodiment, said seed storage protein is moong bean 8S protein.

In some embodiments, said protein content is suspended in a gel.

In some embodiments, said protein content is in the form of a gel.

In one embodiment, said gel comprises an isolated, purifiedcross-linking enzyme.

In some embodiments, said isolated, purified cross-linking enzyme isselected from the group consisting of transglutaminase, lysyl oxidases,and amine oxidases.

In a particular embodiment, said isolated, purified cross-linking enzymeis transglutaminase.

In some embodiments, said protein content has been assembled intofibers.

In particular embodiments, said fibers are arranged isotropically.

In one embodiment, said fibers are asymmetric fibers.

In some embodiments, the muscle tissue replica further comprises one ormore isolated and purified iron-containing proteins.

In some embodiments, said one or more isolated and purifiediron-containing proteins is selected from the group consisting ofhemoglobin, myoglobin, leghemoglobin, non-symbiotic hemoglobin,chlorocruorin, erythrocruorin, neuroglobin, cytoglobin, protoglobin,truncated 2/2 globin, HbN, cyanoglobin, HbO, Glb3, and cytochromes,Hell's gate globin I, bacterial hemoglobins, ciliate myoglobins,flavohemoglobins.

In a particular embodiment, said one or more isolated and purifiediron-containing proteins comprises an amino acid sequence with at least70% homology to SEQ ID NO 1. SEQ ID NO 1:MVAFTEKQDALVSSSFEAFKANIPQYSVVFYTSILEKAPAAKDLFSFLANGVDPTNPKLTGHAEKLFALVRDSAGQLKASGTVVADAALGSVHAQKAVTDPQFVVVKEALLKTIKAAVGDKWSDELSRAWEVAYDELAAAIKKA. In a particular embodiment, said one or more isolatedand purified iron-containing proteins comprises an amino acid sequencewith at least 80% homology to SEQ ID NO 1. In a particular embodiment,said one or more isolated and purified iron-containing proteinscomprises an amino acid sequence with at least 90% homology to SEQ IDNO 1. In a particular embodiment, said one or more isolated and purifiediron-containing proteins comprises an amino acid sequence with at least95% homology to SEQ ID NO 1. In a particular embodiment, said one ormore isolated and purified iron-containing proteins comprises an aminoacid sequence with at least 98% homology to SEQ ID NO 1.

In particular embodiments, the muscle tissue replica comprises a proteincontent, wherein (i) one isolated and purified protein that is not aniron-containing protein accounts for 40-95% of said protein content,(ii) one or more isolated and purified iron-containing proteins accountsfor 1-20% of said protein content, and (iii) one or more cross-linkingreagents accounts for 0.1-35% of said protein content.

In one embodiment, said protein content accounts for 5-50% of saidreplica by weight or by weight/volume.

In one embodiment, said one isolated and purified protein is moong bean8S protein.

In one embodiment, said one or more isolated and purifiediron-containing proteins is leghemoglobin.

In one embodiment, said one or more cross-linking reagents istransglutaminase.

In some embodiments, the muscle tissue replica contains nomethylcellulose, no carrageenan, no caramel color, no konjac flour, nogum arabic, and no acacia gum.

In particular embodiments, the muscle tissue replica additionallycontains less than 1% wheat gluten. In particular embodiments, themuscle tissue replica additionally contains less than 5% wheat gluten.In particular embodiments, the muscle tissue replica additionallycontains less than 10% wheat gluten. In particular embodiments, themuscle tissue replica additionally contains less than 0.1% wheat gluten.

In a more particular embodiment, said muscle tissue replica contains nowheat gluten.

In other particular embodiments, said muscle tissue replica contains nosoy protein isolate.

In other particular embodiments, said muscle tissue replica contains nosoy protein concentrate.

In other particular embodiments, said muscle tissue replica contains nosoy protein.

In a more particular embodiment, said muscle tissue replica containsless than 5% carbohydrates.

In other particular embodiments, said muscle tissue replica contains notofu.

In some embodiments, said muscle tissue replica contains no tofu, and nowheat gluten.

In some embodiments, said muscle tissue replica contains no soy protein,and no wheat gluten.

In some embodiments, said muscle tissue replica contains no animalproducts and less than 5% carbohydrates.

In some embodiments, said muscle tissue replica contains less than 1%cellulose.

In some embodiments, said muscle tissue replica contains less than 5%insoluble carbohydrates.

In some embodiments, said muscle tissue replica contains no soy protein,and less than 1% cellulose.

In some embodiments, said muscle tissue replica contains no soy protein,and less than 5% insoluble carbohydrates.

In some embodiments, said muscle tissue replica contains no wheatgluten, and less than 1% cellulose.

In some embodiments, said muscle tissue replica contains no wheatgluten, and less than 5% insoluble carbohydrates.

In some embodiments, the muscle tissue replica contains nomethylcellulose, no carrageenan, no caramel color, no konjac flour, nogum arabic, and no acacia gum.

In particular embodiments, the meat replica contains less than 1% wheatgluten. In particular embodiments, the meat replica contains less than5% wheat gluten. In particular embodiments, the meat replica containsless than 10% wheat gluten. In particular embodiments, the meat replicacontains less than 0.1% wheat gluten.

In a more particular embodiment, the meat replica contains no wheatgluten.

In other particular embodiments, the meat replica contains no soyprotein isolate.

In other particular embodiments, the meat replica contains no soyprotein concentrate.

In other particular embodiments, the meat replica contains no soyprotein.

In a more particular embodiment, the meat replica contains less than 5%carbohydrates.

In other particular embodiments, the meat replica contains no tofu.

In some embodiments, the meat replica contains no tofu, and no wheatgluten.

In some embodiments, the meat replica contains no soy protein, and nowheat gluten.

In some embodiments, the meat replica contains no animal products andless than 5% carbohydrates.

In some embodiments, the meat replica contains less than 1% cellulose.In some embodiments, the meat replica contains less than 0.1% cellulose.In some embodiments, the meat replica contains less than 10% cellulose.In some embodiments, the meat replica contains less than 5% cellulose.

In some embodiments, the meat replica contains less than 5% insolublecarbohydrates.

In some embodiments, the meat replica contains no soy protein, and lessthan 1% cellulose.

In some embodiments, the meat replica contains no soy protein, and lessthan 5% insoluble carbohydrates.

In some embodiments, the meat replica contains no wheat gluten, and lessthan 1% cellulose.

In some embodiments, the meat replica contains no wheat gluten, and lessthan 5% insoluble carbohydrates.

In another aspect, the invention provides a fat tissue replicacomprising a gelled emulsion, said gelled emulsion comprising a proteinsolution with fat droplets suspended therein.

In some embodiments, said fat droplets are derived from a non-animalsource.

In some embodiments, said fat droplets are comprised of one or moreplant oils.

In some embodiments, said one or more plant oils is selected from thegroup consisting of corn oil, olive oil, soy oil, peanut oil, walnutoil, almond oil, sesame oil, cottonseed oil, rapeseed oil, canola oil,safflower oil, sunflower oil, flax seed oil, algal oil, palm oil, palmkernel oil, coconut oil, babassu oil, shea butter, mango butter, cocoabutter, wheat germ oil, rice bran oil, oils produced by bacteria, algae,archaea or fungi or genetically engineered bacteria, algae, archaea orfungi, triglycerides, monoglycerides, diglycerides, sphingosides,glycolipids, lecithin, lysolecithin, phophatidic acids, lysophosphatidicacids, oleic acid, palmitoleic acid, palmitic acid, myristic acid,lauric acid, myristoleic acid, caproic acid, capric acid, caprylic acid,pelargonic acid, undecanoic acid, linoleic acid, 20:1 eicosanoic acid,arachidonic acid, eicosapentanoic acid, docosohexanoic acid, 18:2conjugated linoleic acid, conjugated oleic acid, or esters of: oleicacid, palmitoleic acid, palmitic acid, myristic acid, lauric acid,myristoleic acid, caproic acid, capric acid, caprylic acid, pelargonicacid, undecanoic acid, linoleic acid, 20:1 eicosanoic acid, arachidonicacid, eicosapentanoic acid, docosohexanoic acid, 18:2 conjugatedlinoleic acid, or conjugated oleic acid, or glycerol esters of oleicacid, palmitoleic acid, palmitic acid, myristic acid, lauric acid,myristoleic acid, caproic acid, capric acid, caprylic acid, pelargonicacid, undecanoic acid, linoleic acid, 20:1 eicosanoic acid, arachidonicacid, eicosapentanoic acid, docosohexanoic acid, 18:2 conjugatedlinoleic acid, or conjugated oleic acid, or triglyceride derivatives ofoleic acid, palmitoleic acid, palmitic acid, myristic acid, lauric acid,myristoleic acid, caproic acid, capric acid, caprylic acid, pelargonicacid, undecanoic acid, linoleic acid, 20:1 eicosanoic acid, arachidonicacid, eicosapentanoic acid, docosohexanoic acid, 18:2 conjugatedlinoleic acid, or conjugated oleic acid.

In one embodiment, said one or more plant oils is rice bran oil orcanola oil.

In some embodiments, said protein solution comprises one or moreisolated, purified proteins.

In some embodiments, said one or more isolated, purified proteinsaccounts for 75% or more of the protein in said protein solution.

In some embodiments, said one or more isolated, purified proteins arederived from a non-animal source.

In some embodiments, said non-animal source is a plant source.

In some embodiments, said non-animal source is a genetically modifiedyeast or bacteria.

In some embodiments, each of said one or more isolated proteins isisolated and purified separately.

In some embodiments, said one or more isolated proteins are selectedfrom the group consisting of hemoglobin, myoglobin, chlorocruorin,erythrocruorin, neuroglobin, cytoglobin, protoglobin, truncated 2/2globin, HbN, cyanoglobin, HbO, Glb3, and cytochromes, Hell's gate globinI, bacterial hemoglobins, ciliate myoglobins, flavohemoglobins,ribosomal proteins, actin, hexokinase, lactate dehydrogenase, fructosebisphosphate aldolase, phosphofructokinases, triose phosphateisomerases, phosphoglycerate kinases, phosphoglycerate mutases,enolases, pyruvate kinases, glyceraldehyde-3-phosphate dehydrogenases,pyruvate decarboxylases, actins, translation elongation factors,ribulose-1,5-bisphosphate carboxylase oxygenase (rubisco),ribulose-1,5-bisphosphate carboxylase oxygenase activase (rubiscoactivase), albumins, glycinins, conglycinins, globulins, vicilins,conalbumin, gliadin, glutelin, gluten, glutenin, hordein, prolamin,phaseolin (protein), proteinoplast, secalin, extensins, triticeaegluten, zein, any seed storage protein, oleosins, caloleosins,steroleosins or other oil body proteins, vegetative storage protein A,vegetative storage protein B, moong seed storage 8S globulin.

In some embodiments, said one or more isolated, purified proteins is analbumin protein, a seed storage protein, or pea globulin protein.

In particular embodiments, said albumin protein is isolated pea albuminprotein.

In some embodiments, said seed storage protein is moong bean 8S protein.

In some embodiments, said gelled emulsion comprises a protein solutioncomprising 1-3 isolated and purified proteins, wherein said solutionaccounts for 30-70% of the volume of said emulsion; a plant oil, whereinsaid plant oil accounts for 30-70% of the volume of said emulsion; andan isolated, purified cross-linking enzyme, wherein said cross-linkingenzyme accounts for 0.5-5% of said emulsion by wt/volume; wherein saidplant oil is emulsified in said protein solution, wherein said emulsionis formed into a gel by said cross-linking enzyme.

In other embodiments said gelled emulsion comprises a protein solutioncomprising 1-3 isolated and purified proteins, wherein said solutionaccounts for 1-30% of the volume of said emulsion; a plant oil, whereinsaid plant oil accounts for 70-99% of the volume of said emulsion; andan isolated, purified cross-linking enzyme, wherein said cross-linkingenzyme accounts for 0.5-5% of said emulsion by wt/volume; wherein saidplant oil is emulsified in said protein solution, wherein said emulsionis formed into a gel by said cross-linking enzyme.

In some embodiments, the fat replica further comprises a cross-linkingenzyme.

In some embodiments, said cross-linking enzyme is transglutaminase.

In some embodiments, one of said 1-3 isolated and purified proteins ismoong bean 8S protein, pea albumin protein, or pea globulin protein.

In particular embodiments, said plant oil is rice bran oil or canolaoil.

In some embodiments, the fat tissue replica contains no methylcellulose,no carrageenan, no caramel color, no konjac flour, no gum arabic, and noacacia gum.

In particular embodiments, the fat tissue replica additionally containsless than 1% wheat gluten.

In a more particular embodiment, said fat tissue replica contains nowheat gluten.

In other particular embodiments, said fat tissue replica contains no soyprotein isolate.

In other particular embodiments, said fat tissue replica contains no soyprotein concentrate.

In other particular embodiments, said fat tissue replica contains no soyprotein.

In a more particular embodiment, said fat tissue replica contains lessthan 5% carbohydrates.

In other particular embodiments, said fat tissue replica contains notofu.

In some embodiments, said fat tissue replica contains no tofu, and nowheat gluten.

In some embodiments, said fat tissue replica contains no soy protein,and no wheat gluten.

In some embodiments, said fat tissue replica contains no animal productsand less than 5% carbohydrates.

In some embodiments, said fat tissue replica contains less than 1%cellulose.

In some embodiments, said fat tissue replica contains less than 5%insoluble carbohydrates.

In some embodiments, said fat tissue replica contains no soy protein,and less than 1% cellulose.

In some embodiments, said fat tissue replica contains no soy protein,and less than 5% insoluble carbohydrates.

In some embodiments, said fat tissue replica contains no wheat gluten,and less than 1% cellulose.

In some embodiments, said fat tissue replica contains no wheat gluten,and less than 5% insoluble carbohydrates.

In another aspect, the invention provides a connective tissue replica,comprising a protein content comprising one or more isolated, purifiedproteins, wherein said protein content has been assembled intostructures approximating the texture and visual appearance of connectivetissue or skin.

In some embodiments, said protein content is derived from non-animalsource.

In some embodiments, said non-animal source is a plant source.

In some embodiments, said non-animal source is a genetically modifiedyeast or bacteria.

In some embodiments, said one or more isolated proteins account for 50%or more of said protein content by weight.

In some embodiments, said one isolated and purified protein accounts for90% or more of said protein content by weight.

In some embodiments, each of said one or more isolated proteins isisolated and purified separately.

In some embodiments, said one or more isolated proteins are selectedfrom the group consisting of hemoglobin, myoglobin, chlorocruorin,erythrocruorin, neuroglobin, cytoglobin, protoglobin, truncated 2/2globin, HbN, cyanoglobin, HbO, Glb3, and cytochromes, Hell's gate globinI, bacterial hemoglobins, ciliate myoglobins, flavohemoglobins,ribosomal proteins, actin, hexokinase, lactate dehydrogenase, fructosebisphosphate aldolase, phosphofructokinases, triose phosphateisomerases, phosphoglycerate kinases, phosphoglycerate mutases,enolases, pyruvate kinases, glyceraldehyde-3-phosphate dehydrogenases,pyruvate decarboxylases, actins, translation elongation factors,ribulose-1,5-bisphosphate carboxylase oxygenase (rubisco),ribulose-1,5-bisphosphate carboxylase oxygenase activase (rubiscoactivase), albumins, glycinins, conglycinins, globulins, vicilins,conalbumin, gliadin, glutelin, gluten, glutenin, hordein, prolamin,phaseolin (protein), proteinoplast, secalin, extensins, triticeaegluten, zein, any seed storage protein, oleosins, caloleosins,steroleosins or other oil body proteins, vegetative storage protein A,vegetative storage protein B, moong seed storage 8S globulin.

In some embodiments, said one or more isolated and purified proteins isa prolamin family protein.

In some embodiments, said one or more isolated and purified proteins iszein.

In some embodiments, said protein content is suspended in a gel.

In some embodiments, said gel comprises an isolated, purifiedcross-linking enzyme.

In some embodiments, said isolated, purified cross-linking enzyme isselected from the group consisting of transglutaminase, lysyl oxidases,and amine oxidases.

In some embodiments, said isolated, purified cross-linking enzyme istransglutaminase.

In some embodiments, said protein content is formed into a fiber.

In some embodiments, said fiber is produced by an extrusion process.

In some embodiments, said fiber is stabilized by protein crosslinks.

In some embodiments, fiber contains an isolated, purified cross-linkingenzyme.

In some embodiments, said isolated, purified cross-linking enzyme isselected from the group consisting of transglutaminase, lysyl oxidases,and amine oxidases.

In some embodiments, said isolated, purified cross-linking enzyme istransglutaminase.

In another aspect, the invention provides a meat substitute product,comprising a muscle replica; a fat tissue replica; and a connectivetissue replica; wherein said muscle replica, fat tissue replica, and/orconnective tissue replica are assembled in a manner that approximatesthe physical organization of meat.

In some embodiments, the meat substitute product comprises two or moreof said muscle replica, fat tissue replica, and connective tissuereplica.

In some embodiments of the meat substitute product, said muscle replicaaccounts for 40-90% of said product by weight, said fat tissue replicaaccounts for 1-60% of said product by weight, and said connective tissuereplica accounts for 1-30% of said product by weight.

In some embodiments, the meat substitute product comprises 60-90% water;5-30% protein content; and 1-20% of a fat or fat replica; wherein saidprotein content comprises one or more isolated, purified plant proteins.

In some embodiments, said protein content is derived from non-animalsource.

In some embodiments, non-animal source is a plant source.

In some embodiments, said non-animal source is a genetically modifiedyeast or bacteria.

In some embodiments, 50% or more of said protein content by weight areone or more isolated purified proteins.

In some embodiments, each of said one or more isolated proteins isisolated and purified separately from different plant species.

In some embodiments, one or more of said isolated proteins is selectedfrom the group consisting of: hemoglobin, myoglobin, chlorocruorin,erythrocruorin, neuroglobin, cytoglobin, protoglobin, truncated 2/2globin, HbN, cyanoglobin, HbO, Glb3, and cytochromes, Hell's gate globinI, bacterial hemoglobins, ciliate myoglobins, flavohemoglobins,ribosomal proteins, actin, hexokinase, lactate dehydrogenase, fructosebisphosphate aldolase, phosphofructokinases, triose phosphateisomerases, phosphoglycerate kinases, phosphoglycerate mutases,enolases, pyruvate kinases, glyceraldehyde-3-phosphate dehydrogenases,pyruvate decarboxylases, actins, translation elongation factors,ribulose-1,5-bisphosphate carboxylase oxygenase (rubisco),ribulose-1,5-bisphosphate carboxylase oxygenase activase (rubiscoactivase), albumins, glycinins, conglycinins, globulins, vicilins,conalbumin, gliadin, glutelin, gluten, glutenin, hordein, prolamin,phaseolin (protein), proteinoplast, secalin, extensins, triticeaegluten, zein, any seed storage protein, oleosins, caloleosins,steroleosins or other oil body proteins, vegetative storage protein A,vegetative storage protein B, moong seed storage 8S globulin.

In some embodiments, the meat substitute product further comprises oneor more isolated and purified iron-containing proteins.

In some embodiments, said one or more isolated and purifiediron-containing proteins is selected from the group consisting ofhemoglobin, myoglobin, leghemoglobin, non-symbiotic hemoglobin,chlorocruorin, erythrocruorin, neuroglobin, cytoglobin, protoglobin,truncated 2/2 globin, HbN, cyanoglobin, HbO, Glb3, and Hell's gateglobin I, bacterial hemoglobins, ciliate myoglobins, flavohemoglobins.In some embodiments, said iron-containing protein comprises an aminoacid sequence with at least 70% homology to SEQ ID NO 1. [SEQ ID NO 1:MVAFTEKQDALVSSSFEAFKANIPQYSVVFYTSILEKAPAAKDLFSFLANGVDPTNPKLTGHAEKLFALVRDSAGQLKASGTVVADAALGSVHAQKAVTDPQFVVVKEALLKTIKAAVGDKWSDELSRAWEVAYDELAAAIKKA]

In some embodiments, the meat substitute product contains nomethylcellulose, no carrageenan, no caramel color, no konjac flour, nogum arabic, and no acacia gum.

In particular embodiments, the meat substitute product additionallycontains less than 1% wheat gluten.

In a more particular embodiment, said meat substitute product containsno wheat gluten.

In other particular embodiments, said meat substitute product containsno soy protein isolate.

In other particular embodiments, said meat substitute product containsno soy protein concentrate.

In other particular embodiments, said meat substitute product containsno soy protein.

In a more particular embodiment, said meat substitute product containsless than 5% carbohydrates.

In other particular embodiments, said meat substitute product containsno tofu.

In some embodiments, said meat substitute product contains no tofu, andno wheat gluten.

In some embodiments, said meat substitute product contains no soyprotein, and no wheat gluten.

In some embodiments, said meat substitute product contains no animalproducts and less than 5% carbohydrates.

In some embodiments, said meat substitute product contains less than 1%cellulose.

In some embodiments, said meat substitute product contains less than 5%insoluble carbohydrates.

In some embodiments, said meat substitute product contains no soyprotein, and less than 1% cellulose.

In some embodiments, said meat substitute product contains no soyprotein, and less than 5% insoluble carbohydrates.

In some embodiments, said meat substitute product contains no wheatgluten, and less than 1% cellulose.

In some embodiments, said meat substitute product contains no wheatgluten, and less than 5% insoluble carbohydrates.

In another aspect, the invention provides a food product comprising oneor more isolated, purified iron-containing proteins, wherein said foodproduct is configured for consumption by an animal.

In some embodiments, said one or more isolated, purified iron-containingproteins is derived from a non-animal source.

In some embodiments, said non-animal source is a plant source.

In some embodiments, said plant source comprises one or more plants ofthe legume family.

In some embodiments, said one or more plants of the legume family is asoy or pea plant.

In some embodiments, said non-animal source is a genetically modifiedyeast or bacteria.

In some embodiments, said iron-containing protein is selected from thegroup consisting of hemoglobin, myoglobin, leghemoglobin, non-symbiotichemoglobin, chlorocruorin, erythrocruorin, neuroglobin, cytoglobin,protoglobin, truncated 2/2 globin, HbN, cyanoglobin, HbO, Glb3, andcytochromes, Hell's gate globin I, bacterial hemoglobins, ciliatemyoglobins, flavohemoglobins.

In one embodiment, said iron-containing protein comprises an amino acidsequence with at least 70% homology to SEQ ID NO 1. [SEQ ID NO 1:MVAFTEKQDALVSSSFEAFKANIPQYSVVFYTSILEKAPAAKDLF SFLANGVDPTNPKLTGHAEKLFALVRDSAGQLKASGTVVADAALGSVHAQKAVTDPQFVVVKEALLKTIKAAVGDKWSDELSRAWEVAYDELAAAIKKA]

In some embodiments, the food product contains no methylcellulose, nocarrageenan, no caramel color, no konjac flour, no gum arabic, and noacacia gum.

In particular embodiments, the food product additionally contains lessthan 1% wheat gluten.

In a more particular embodiment, said food product contains no wheatgluten.

In other particular embodiments, said food product contains no soyprotein isolate.

In other particular embodiments, said food product contains no soyprotein concentrate.

In other particular embodiments, said food product contains no soyprotein.

In a more particular embodiment, said food product contains less than 5%carbohydrates.

In other particular embodiments, said food product contains no tofu.

In some embodiments, said food product contains no tofu, and no wheatgluten.

In some embodiments, said food product contains no soy protein, and nowheat gluten.

In some embodiments, said food product contains no animal products andless than 5% carbohydrates.

In some embodiments, said food product contains less than 1% cellulose.

In some embodiments, said food product contains less than 5% insolublecarbohydrates.

In some embodiments, said food product contains no soy protein, and lessthan 1% cellulose.

In some embodiments, said food product contains no soy protein, and lessthan 5% insoluble carbohydrates.

In some embodiments, said food product contains no wheat gluten, andless than 1% cellulose.

In some embodiments, said food product contains no wheat gluten, andless than 5% insoluble carbohydrates.

In another aspect, the invention provides a method of making a meatsubstitute composition, comprising isolating and purifying one or moreproteins; and assembling said one or more proteins into a physicalorganization that approximates the physical organization of meat.

In another aspect, the invention provides a method of making a muscletissue replica, comprising isolating and purifying one or more proteins;and assembling said one or more proteins into a physical organizationthat approximates the physical organization of skeletal muscle.

In another aspect, the invention provides a method of making a fattissue replica, comprising isolating and purifying one or more proteins;preparing a solution comprising one or more proteins; emulsifying one ormore fats in said solution; and stabilizing said solution into a gelledemulsification with one or more cross-linking reagents.

In another aspect, the invention provides a method of making aconnective tissue replica, comprising isolating and purifying one ormore proteins; and precipitating said one or more proteins, wherein saidprecipitating results in said one or more proteins forming physicalstructures approximating the physical organization of connective tissue.

In some embodiments, said precipitating comprises solubilizing said oneor more proteins in a first solution; and extruding said first solutioninto a second solution, wherein said one or more proteins is insolublein said second solution, wherein said extruding induces precipitation ofsaid one or more proteins.

In another aspect, the invention provides a food product comprising oneor more isolated, purified iron-containing proteins, wherein said foodproduct is configured for consumption by an animal.

In another aspect, the invention provides a food product comprising oneor more isolated, purified iron-containing proteins, wherein said foodproduct is configured for consumption by humans.

In another aspect, the invention provides a food product comprising oneor more isolated, purified iron-containing proteins, wherein said foodproduct is configured for consumption by an animal. In another aspect,the invention provides a food product comprising one or more isolated,purified iron-containing proteins, wherein said food product isconfigured for consumption by humans.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 depicts a portion of the root of a pea plant (Pisum sativum) withthe root nodules sliced open to demonstrate the red color conferred byleghemoglobin contained therein. The sliced open rood nodule appearsred.

FIG. 2 depicts leghemoglobin isolated from 1 oz of pea roots. The redcolor commonly attributed to meat is evident in the color photo.

FIG. 3 shows that leghemoglobins from different species are homologs andhave similar color properties. In FIG. 3, panel A shows an SDS_PAGE gelsof lysed root-nodules of three legume plant species (1) Fava bean (2)English Pea (3) Soybean. Arrows mark respective leghemoglobins. Panel Bshows the similarity of UV-VIS spectral profile of leghemoglobins fromtwo different plant species (Favabean and Soybean).

FIG. 4 shows a comparison of reduced (heme iron 2+) and oxidized (hemeiron 3+) soybean leghemoglobin (FIG. 4 panel A) and equine heart musclemyoglobin (FIG. 4 panel B) showing similarity of UV-VIS absorptionprofiles of two proteins. We purified soybean leghemoglobin from soybeanroot-nodules using here described protocol. Purified equine myoglobinwas purchased from SigmaAldrich. Soybean leghemoglobin (FIG. 4 panel A)and equine myoglobin (FIG. 4 panel B) were reduced with 1 mm sodiumhydrosulfite. Shown are UV-VIS absorption spectra of heme Fe3+(blueline—the higher peak in FIGS. 4 and 5) and heme Fe2+(red line) ofsoybean leghemoglobin (FIG. 4 panel A) and equine myoglobin (FIG. 4panel B). Insets show a zoom-in of UV-VIS spectra in 450 nm to 700 nmregion. (FIG. 4 panel C) Images of 10 ul liquid droplet of a 40 mg/mlsolution of soybean leghemoglobin in the heme-Fe3+ state (left droplet)showing characteristic rusty red color and a 40 mg/ml solution ofsoybean leghemoglobin solution in the heme-Fe2+ state (right droplet)showing characteristic red color of and (right image) correspondingsamples of equine myoglobin.

FIG. 5 depicts examples of successful reduction of leghemoglobin hemeiron with sodium hydrosulfite and titanium citrate. In FIG. 5 the UV-VISspectrogram of purified soybean leghemoglobin in which the heme iron isin the oxidized (+3) state is represented by the blue curves in eachpanel (the blue curves have the higher peaks on the main graphs). Thered curves in each panel represent the UV-VIS spectra of the sameleghemoglobin species after reduction to the (+2) state (red lines) byaddition of (Panel A) 1 mM sodium hydrosulfite or (Panel B) 0.24% (wt/v)titanium citrate in 20 mM potassium phosphate pH 7.3, 100 mM sodiumchloride buffer. The Insets show a zoom-in of UV-VIS spectra in 450-700nm region. For this example, leghemoglobin was purified from soybeanroot nodules using 60/90% ammonium sulfate fractionation and exchangedinto 20 mM potassium phosphate pH 7.4, 100 mM sodium chloride buffer.Sodium hydrosulfite stock solution was prepared by dissolving 100 mMsodium hydrosulfite in 1 mM sodium hydroxide in water. Titanium citratestock solution was prepared from 20% (wt/v) Ti-chloride in hydrochloricacid by mixing it with 0.2M sodium citrate (1:10 v/v). pH was adjustedusing sodium carbonate to pH 7.0.

FIG. 6 depicts an example of the leghemoglobin purification flow fromsoybean root nodules. The figure shows SDS-PAGE fractionation ofdifferent soybean leghemoglobin purification steps (Lane 1) Soybeanroot-nodule lysate; (Lane 2) Soybean root-nodule lysate purified by60/90% (wt/v) ammonium sulfate fractionation. Shown is the proteincontent of 90% ammonium sulfate fractionated protein pellet resuspendedin 20 mm potassium phosphate pH 7.4, 100 mM sodium chloride, 1 mM EDTAbuffer; Proteins from 90% ammonium sulfate pellet were further purifiedby anion-exchange chromatography (FFQ GE Healthcare) in 20 mM potassiumphosphate ph 7.4, 100 mM sodium chloride. Leghemoglobin collected in theflowthrough is shown in Lane 3. Anion-exchange flowthrough wasfractionated using size-exclusion chromatography (Sephacryl S-100 GEHealthcare) and resulting leghemoglobin fraction is shown in Lane 4.Leghemoglobin content at different purification steps was determined bydetermining the fraction of leghemoglobin band on SDS-PAGE gel in arespective sample using ImageDoc analysis software (BioRad). Purity(partial abundance) of leghemoglobin at respective steps in thepurification steps was: lysate: 32.7% (lane 1), 60/90% (wt/v) ammoniumsulfate fractionation 78% (lane 2), anion-exchange chromatography ˜83%(lane 3), and size-exclusion chromatography to ˜95% (lane 4).

FIG. 7 shows stained SDS-PAGE gel analysis of (A) soybean leghemoglobinexpressed and purified using recombinant protein technology and (B)soybean leghemoglobin purified from soybean root nodules. (A)Recombinant Soybean leghemoglobin A carrying His-tag and TEV proteaseHis-tag removal site was expressed in E. coli BL21 strain and purifiedusing His-tag affinity chromatography (Talon resin, CloneTech). The leftlane contains molecular weight standards, the right lane containspurified recombinant soybean leghemoglobin A (arrow). Expected molecularweight of the recombinant soybean leghemoglobin A is 17.1 kDa. (B)SDS-PAGE gel of purified Soybean leghemoglobin from root nodules. Theleft lane contains molecular weight standards, the right lane containspurified soybean leghemoglobin A (arrow). Mass spectrometry analysis ofpurified material determined that all four soybean leghemoglobinisoforms are present, and are full length (data not shown). Expectedmolecular weights (MW) of soybean leghemoglobin isoforms range fromMW15.4 to 15.8 kDa.

FIG. 8 shows an example of 6 cubes of a commercial meat analog (Quornchicken analog), about 1 cm on a side, 4 of which (Left and lower right)have been soaked in a solution of about 10 mg/ml soybean leghemoglobinin 20 mM Potassium phosphate pH 7.4 and 100 mM NaCl; the remaining two(Upper right) were soaked in the same buffer without leghemoglobin. Adeep pink color of the leghemoglobin-infused cubes is apparent in colorphotos contrasting the pale tan color of the un-infused cubes.

FIG. 9 shows the 4 leghemoglobin-infused cubes of Quorn chicken analogin the process of cooking in a pan at 350° C. The two lower cubes havebeen turned over to expose the grilled surface, which has turned brown.In the upper two cubes that the heated portion has turned grey-brown,while the cooler top surface retains its pink color. In some embodimentsthe consumable is injected with a heme containing solution, for instancea leghemoglobin solution, until the consumable is the color of uncookedmeat.

FIG. 10 depicts 43 ml of moong bean protein solution (150 mg/ml indialysis buffer) were mixed with 37 ml of leghemoglobin solution (46.5mg/ml leghemoglobin and 20 mg/ml of other soybean root nodule protein)in 20 mM potassium phosphate, 100 mM NaCl, pH 7.3). 20 ml oftransglutaminase solution (20% w/w) were added, solutions thoroughlymixed, divided into two 50 ml Falcon tubes and incubated overnight atroom temperature. Final protein concentrations were 65 mg/ml for moongbean protein, 18 mg/ml of leghemoglobin, 91 mg/ml total protein.

FIG. 11 depicts “White” muscle analog prepared by mixing 43 ml moongbean protein solution (150 mg/ml) with 45 ml of 11.7 mg/ml solution ofleghemoglobin and 0.8% (wt/v) of transglutaminase solution. Finalprotein concentrations were 63 mg/ml for moong bean protein, 5.2 mg/mlof leghemoglobin, 68 mg/ml total protein.

FIG. 12 depicts a fat tissue analog based on moong beans and prepared ineppendorf tubes formed an opaque gel of off-white color, smooth uniformtexture, with no visible discernible liquid that was not incorporatedinto the gel. The gel was freely standing, elastic and springy. The gelhas a slight, pleasant aroma and a mild and pleasant flavor. The tasteis mildly salty.

FIG. 13 depicts at tissue analog based on pea globulin and prepared ineppendorf tubes very similar to moong bean-based fat analog, except thatit gave up a little of oil upon compression.

FIG. 14 shows connective-tissue analog strands that were created using a1:3 ratio in 70% ethanol, loaded into a syringe with a 23 gauge needle(ID 0.337 mm) The solution was slowly extruded from the bottom of a 5inch-high vessel into an excess of 5 M NaCl solution. The ethanol-zeinsolution being less dense than the NaCl solution, floated upward,drawing out a fibrous stand of solidifying zein. The NaCl was constantlystirred as the strands began to develop to assist in the strandlengthening. The strands bunch together and become a hard, dense mass.

FIG. 15 depicts a ground beef prototype patty was made by combining 62%(wt/wt) muscle analog (62% (wt/wt) “dark muscle analog” and 38% (wt/wt)“white muscle analog”), 29% (wt/wt) fat tissue analog (from pea globulinand canola oil), 5% (wt/wt) connective tissue analog (FIG. 15 panel A).A ground beef prototype patty was made by combining 62% muscle analog(62% “dark muscle analog” and 38% “white muscle analog), 29% fat tissueanalog (from moong bean seed 8S protein and rice bran oil), 5%connective tissue analog (FIG. 15 panel B). A ground beef prototypepatty was made by combining 71% (wt/wt) muscle tissue analog (composedof 60% “white” muscle analog, 40% “dark” muscle analog), 23% fat tissue(from pea seed globulin proteins and canola oil) (FIG. 15 panel C). Aground beef prototype patty was made by combining 67% “White” muscleanalog, with 28% fat tissue analog (from pea globulins and rice branoil), (FIG. 15, panel D)

FIG. 16 depicts a ground beef patty analog was made by combining 62%(wt/wt) muscle tissue analog (62% (wt/wt) “dark muscle analog” and 38%(wt/wt) “muscle analog”), 29% (wt/wt) fat tissue analog (from peaglobulin and canola oil), 5% (wt/wt) connective tissue analog. The panelon the left shows the patty before cooking and the panel on the rightshows the same patty after cooking for about 2 minutes. Observersdescribed the aroma of the cooking ground beef replica as distinctly“beefy”.

DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions for the production of consumables are describedherein. The consumables can be for animal consumption. For example theconsumable can be food fit for human consumption. The consumable can beapproved by suitable regulatory authorities. The consumables can be soldin grocery stores or prepared in restaurants, schools, hospitals,military facilities, prisons, shelters, long-term care facilities,similar to already existing human foods. The consumables could also befood for domestic animals. For instance, dog food could be producedaccording to the present inventions. The consumables may also be foodfor wild animals. For instance, the consumables could be provided tonon-domesticated predatory animals.

The consumables of the present invention can compete with, supplement orreplace animal based foods. For instance the consumables can be meatreplicas made entirely from plant sources. The consumables can be madeto mimic the cut or appearance of meat as it is currently sold. Forinstance a consumable may be visually similar to or indistinguishablefrom ground beef or a particular cut of beef. Alternatively, theconsumables can be made with a unique look or appearance. For instancethe consumable could contain patterns or lettering that is based uponthe structure of the consumable. In some instances the consumables looklike traditional meat products after they are prepared. For example aconsumable may be produced which is larger than a traditional cut ofbeef but which, after the consumable is sliced and cooked appears thesame as a traditional cooked meet. In some embodiments the consumablemay resemble a traditional meat shape in two dimensions, but not in athird. For example the consumable may resemble a cut of meat in twodimensions (for example when viewed from the top), but may be muchlonger (or thinker) than the traditional cut. So in some embodiments acomposition that can be cut repeatedly into traditionally meat shapedproducts is provided.

The consumable may be made entirely from plant based sources. In someinstances the consumable can be made from organic sources. Theconsumables may also be made from a combination of plant based sourcesand animal based sources. For instance, the consumable may be a groundbeef product supplemented with plant based products of the invention.

The consumables can be made from local products. For instance theconsumables can be made from plants grown within a certain radius of theeventual consumer. That radius could be 1, 10, 100, or 1000 miles forexample. So, in some embodiments, the invention provides a method forproducing a meat replica which does not contain products which have beenshipped over 1, 10, 100, or 1000 miles prior to producing the meatreplica.

The present invention provides methods for producing consistentproperties from the consumables when they are produced from varioussources. So, for example, a plant based meat replica produced from localplants in Iowa, USA, will have substantially similar taste, odor, andtexture as a plant based meat replica produced from local plants inLorraine, France. This consistency allows for methods for advertisinglocally grown foods with consistent properties. The consistency canarise from the concentration or purification of similar components atdifferent locations. These components can be combined in predeterminedratios to insure consistency. In some embodiments a high degree ofcharacteristic consistency is possible using components (e.g. isolatedor concentrated proteins and fats) which come from the same plantspecies. In some embodiments a high degree of characteristic consistencyis possible using components (e.g. isolated or concentrated proteins andfats) which come from the different plant species. In some embodimentsthe same proteins can be isolated from different plant species. In someembodiments the invention provides for a method comprising isolatingsimilar plant constituents from plant sources in different locations,assembling in both locations compositions provided herein, and sellingthe compositions, wherein the compositions assembled and sold atdifferent the geographic locations have consistent physical and chemicalproperties. In some embodiments the isolated constituents are fromdifferent plant populations in different locations. In some embodimentsone or more of the isolated constituents are shipped to the separategeographic locations.

The consumables may require fewer resources to produce than consumablesproduced from domesticated animals. Accordingly, the present inventionprovides for meat replicates which require less water or energy toproduce than meat. For example a consumable can require less than about10, 50, 100, 200, 300, 500, or 1000 gallons of water per pound ofconsumable. For comparison beef can require over 2000 gallons of waterper pound of meat.

The consumable may require less land are to produce than a meat productwith similar protein content. For example the consumable may require 30%or less of the land area required to produce a meat product with similarprotein content.

The consumable may have health benefits compared to an animal product itreplaces in the diet. For example it may have less cholesterol or lowerlevels of saturated fats than comparable meat products.

The consumable may have animal welfare benefits compared to an animalproduct it replaces in the diet. For instance it may be produced withoutrequiring confinement, forced feeding, premature weaning, disruption ofmaternal-offspring interactions, or slaughter of animals for their meat.

The consumable may have a smaller “carbon footprint” than the meatproducts they replace. For example the consumable may result in netgreenhouse gas emissions of 1%, 5%, 10%, 25%, 50% or 75% of thegreenhouse gas emissions attributable to the animal product it replaces.

The consumable may provide alternatives to animal products orcombinations of animal products whose consumption is forbidden byreligious beliefs. For example, the consumable may be a kosher porkchop.

The consumable can also be shipped in components and produced orassembled at a different location. When available, local components canbe used for production of the consumable. These can be supplemented withcomponents which are not locally available. This allows for methods ofproducing consumables, for instance meat replicates, using less energyin shipment than is required for meat. For example, local water can beused in combination with a kit which provides other components of theconsumable. Using local water will reduce shipping weight therebyreducing cost and environmental impact.

The consumables can be produced or assembled wholly or in part in areaswhere animal farming is not practical or is not allowed. The consumablecan be produced or assembled within an urban environment. For example akit may be provided to a user to enable the user to produce theconsumable. The user could use local water or use plants from a rooftopgarden, for instance in Shanghai. In another example, the consumablescould be produced aboard a space craft, space station, or lunar base.Accordingly, the present invention provides methods and systems for theproduction of meat replicas for use in space travel or for training forthe same. For instance the present invention could be used in earthbased training for space travel. The consumables could also be producedon an island or upon a manmade platform at sea where the keeping oflivestock is difficult or prohibited.

The consumables are, in some embodiments, designed to replicate theexperience of eating meat. The look, texture, and taste of theconsumable can be such that it is similar or indistinguishable frommeat. The invention therefore provides in certain embodiments methodsfor determining whether an animal or human can distinguish theconsumable from meat.

One method to determine whether the consumable is comparable to meat isto a) define the properties of meat and b) determine whether theconsumable has similar properties. Properties of meat that can be testedinclude mechanical properties such as hardness, cohesiveness,brittleness, chewiness, gumminess, viscosity, elasticity, andadhesiveness. Properties of meat that can be tested also includegeometric properties such as particle size and shape, and particle shapeand orientation. Additional properties can include moisture content andfat content. These properties can be described using terms such as“soft,” “firm” or “hard” describe hardness; “crumbly,” “crunchy,”“brittle,” “chewy,” “tender,” “tough,” “short,” “mealy,” “pasty,” or“gummy,” to describe cohesiveness; “thin” or “viscous” to describeviscosity; “plastic” or “elastic” to describe elasticity; “sticky,”“tacky” or “gooey” to describe adhesiveness; “gritty,” “grainy” or“course” to describe particle shape and size; “fibrous,” “cellular” or“crystalline” to describe particle shape and orientation, “dry,”“moist,” “wet,” or “watery” to describe moisture content; or “oily” or“greasy” to describe fat content. So, in one embodiment a group ofpeople can be asked to rate a certain meat, for instance ground beef,according to properties which describe the meat. These ratings can beused as an indication of the properties of the meat. The consumables ofthe present invention can then be compared to the meat to determine howsimilar the consumable is to the meat. In some instances the propertiesof the consumables are then altered to make the consumable more similarto the meat. So, in some embodiments, the consumable is rated similar tomeat according to human evaluation. In some embodiments the consumableis indistinguishable from real meat to a human.

In some embodiments, subjects asked to identify the consumable identifyit as a form of meat. In some embodiments one property of thecompositions of the invention is that an animal, for example a human,will identify the composition as a meat. In some embodiments the humanidentifies the composition of the invention as having propertiesequivalent to meat. In some embodiments one or more properties of meatare equivalent according to a human's perception. Such propertiesinclude the properties that can be tested. In some embodiments a humanidentifies a consumable of the present invention as more meat like thanmeat substitutes found in the art.

In embodiments an experiment can demonstrate that consumable sacceptable to consumers. A panel can be used to screen a variety ofconsumables described herein. A number of human panelists can testedmultiple consumable samples, namely, natural meats vs. the consumablecompositions described herein. Variables such as fat content can bestandardized for example to 20% fat using lean and fat meat mixes. Fatcontent can be determined using the Babcock for meat method (S. S.Nielson, Introduction to the Chemical Analysis of Foods (Jones &Bartlett Publishers, Boston, 1994)). Mixtures of ground beef andconsumables of the invention prepared according to the proceduredescribed herein can be formulated.

Panelists can be served samples in booths, under red lights or underwhite light, in an open consumer panel. Samples can be assigned randomthree-digit numbers and rotated in ballot position to prevent bias.Panelists can be asked to evaluate samples for tenderness, juiciness,texture, flavor, and overall acceptability using a hedonic scale from1=dislike extremely, to 9=like extremely, with a median of 5=neitherlike nor, dislike. Panelists can be encouraged to rinse their mouthswith water between samples, and given opportunity to comment on eachsample.

The results of this experiment can indicate significant differences(p<0.05) or similarities between the traditional meats and thecompositions of the invention.

These results will demonstrate that the compositions of the inventionare judged as acceptably equivalent to real meat products. Additionallythese results can demonstrate that compositions of the invention arepreferred by panelist over other commercially available meatsubstitutes. So, in some embodiments the present invention provides forconsumables that are significantly similar to traditional meats.

Consumables of the invention can also have similar physicalcharacteristics as traditional meat. In one embodiment the forcerequired to pierce a 1 inch thick structure (e.g. a patty) made of aconsumable of the invention with a fixed diameter steel rod is notsignificantly different than the force required to pierce a 1 inch thicksimilar meat structure (e.g. a ground beef patty) with a similar fixeddiameter steel rod. Accordingly, the invention provides for consumableswith similar physical strength characteristics to meat.

In some embodiments composition of the invention have a similar cookloss characteristic as meat. In one embodiment a consumable of theinvention with a similar fat and protein content as ground beef has thesame reduction in size when cooked as real ground beef Similarsimilarities in size loss profiles can be achieved for variouscompositions of consumables described herein matched to various meats.

In some embodiments the consumable is compared to real meat based uponolfactometer readings. In various embodiments the olfactometer can beused to assess odor concentration and odor thresholds, odorsuprathresholds with comparison to a reference gas, hedonic scale scoresto determine the degree of appreciation, or relative intensity of odors.In some embodiments the olfactometer allows the training and automaticevaluation of expert panels. So in some embodiments the consumable is aproduct that causes similar or identical olfactometer readings. In someembodiments the similarity is sufficient to be beyond the detectionthreshold of human perception.

Gas chromatography-mass spectrometry (GCMS) is a method that combinesthe features of gas-liquid chromatography and mass spectrometry toseparate and identify different substances within a test sample. GCMScan, in some embodiments, be used to evaluate the properties of aconsumable. For example volatile chemicals can be isolated from the headspace around meat. These chemicals can be identified using GCMS. Aprofile of the volatile chemicals in the headspace around meat isthereby created. In some instances each peak of the GCMS can be furtherevaluated. For instance, a human could rate the experience of smellingthe chemical responsible for a certain peak. This information could beused to further refine the profile. GCMS could then be used to evaluatethe properties of the consumable. The GCMS profile could be used torefine the consumable.

Characteristic flavor and fragrance components are mostly producedduring the cooking process by chemical reactions molecules includingamino acids, fats and sugars which are found in plants as well as meat.Therefore in some embodiments the consumable is tested for similarity tomeat during or after cooking. In some embodiments human ratings, humanevaluation, olfactometer readings, or GCMS measurements, or combinationsthereof, are used to create an olfactory map of cooked meat. Similarly,an olfactory map of the consumable, for instance a meat replica, can becreated. These maps can be compared to assess how similar the cookedconsumable it so meat. In some embodiments the olfactory map of theconsumable during or after cooking is similar to or indistinguishablefrom that of cooked or cooking meat. In some embodiments the similarityis sufficient to be beyond the detection threshold of human perception.

In one aspect, the invention provides a meat substitute product(alternatively referred to herein as “consumable”) that is substantiallyor entirely composed of ingredients derived from non-animal sources, yetrecapitulates key features associated with the cooking and consumptionof an equivalent meat product derived from animals. The equivalent meatproduct can be a white meat or a dark meat. The equivalent meat productcan be derived from any animal. Non-limiting examples of animals used toderive the equivalent meat product include farmed animals such as, e.g.,cattle, sheep, pig, chicken, turkey, goose, duck, horse, dog or gameanimals (whether wild or farmed) such as, e.g., rabbit, deer, bison,buffalo, boar, snake, pheasant, quail, bear, elk, antelope, pigeon,dove, grouse, fox, wild pig, goat, kangaroo, emu, alligator, crocodile,turtle, groundhog, marmot, possum, partridge, squirrel, raccoon, whale,seal, ostrich, capybara, nutria, guinea pig, rat, mice, vole, anyvariety of insect or other arthropod, seafood such as, e.g, fish, crab,lobster, oyster, muscle, scallop, abalone, squid, octopus, sea urchin,tunicate and others. Many meat products are typically derived fromskeletal muscle of an animal but it is understood that meat can alsocome from other muscles or organs of the animal. In some embodiments,the equivalent meat product is a cut of meat derived from skeletalmuscle. In other embodiments, the equivalent meat product is an organsuch as, e.g., a kidney, heart, liver, gallbladder, intestine, stomach,bone marrow, brain, thymus, lung, tongue. Accordingly, in someembodiments the compositions of the present invention are consumablessimilar to skeletal muscle or organs.

In some aspects, the present invention provides meat substitute productscomprising one or more of a first composition comprising a muscle tissuereplica, a second composition comprising an adipose tissue replica,and/or a third composition comprising a connective tissue replica,wherein the one or more compositions are combined in a manner thatrecapitulates the physical organization of meat. In other aspects, thepresent invention provides compositions for a muscle tissue replica(herein referred to as “muscle replica”), an adipose tissue replica(herein referred to as “fat replica”), and a connective tissue replica(herein referred to as “connective tissue replica”). In someembodiments, the compositions and meat substitute products areprincipally or entirely composed of ingredients derived from non-animalsources. In alternative embodiments, the muscle, fat, and/or connectivetissue replica, or the meat substitute products comprising one or moreof said replicas, are partially derived from animal sources butsupplemented with ingredients derived from non-animal sources. In yetother alternative embodiments, the invention provides meat productssubstantially derived from animal sources but which are supplementedwith one or more of a muscle tissue replica, a fat replica, and/or aconnective tissue replica, wherein said replicas are derivedsubstantially or entirely from non-animal sources. A non-limitingexample of such a meat product is an ultra-lean ground beef productsupplemented with a non-animal derived fat replica which improvestexture and mouthfeel while preserving the health benefits of aconsumable low in animal fat. Such alternative embodiments result inproducts with properties that more closely recapitulate key featuresassociated with preparing and consuming meat but which are less costlyand associated with a lesser environmental impact, less animal welfareimpact, or improved health benefits for the consumer.

The physical organization of the meat substitute product can bemanipulated by controlling the localization, organization, assembly, ororientation of the muscle, fat, and/or connective tissue replicasdescribed herein. In some embodiments the product is designed in such away that the replicas described herein are associated with one anotheras in meat. In some embodiments the consumable is designed so that aftercooking the replicas described herein are associated with one another asin cooked meat. In some embodiments, one or more of the muscle, fat,and/or connective tissue replicas are combined in a manner thatrecapitulate the physical organization of different cuts or preparationsof meat. In an example embodiment, the replicas are combined in a mannerthat approximates the physical organization of natural ground meat. Inother embodiments, the replicas are combined in a manner thatapproximates different cuts of beef, such as, e.g., ribeye, filetmignon, London broil, among others.

Proteins and Protein Sources

In some embodiments, any of the meat substitute products, muscle tissuereplica, fat replica, or connective tissue replica, comprise one or moreisolated, purified proteins. In some embodiments, the meat substituteproducts are comprised of one or more of a muscle replica, a fatreplica, and/or connective tissue replica which comprise one or moreisolated, purified proteins. In other embodiments, the muscle replica,fat replica, and/or connective tissue replica comprises one or moreisolated, purified proteins. In some embodiments, about 0.1%, 0.2%,0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or moreof the protein component is comprised of one or more isolated, purifiedproteins. For the purposes of this document, “purified protein” willrefer to a preparation in which the cumulative abundance by mass ofprotein components other than the specified protein, which can be asingle monomeric or multimeric protein species, is reduced by a factorof 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 50 or more,100 or more or 1000 or more relative to the source material from whichthe specified protein was isolated.

In some embodiments, the one or more isolated, purified proteins arederived from non-animal sources. Non-limiting examples of non-animalsources include plants, funghi, bacteria, archaea, genetically modifiedorganisms such as genetically modified bacteria or yeast, chemical or invitro synthesis. In particular embodiments, the one or more isolated,purified proteins are derived from plant sources. Non-limiting examplesof plant sources include grains such as, e.g., corn, maize, rice, wheat,barley, rye, triticale, teff, oilseeds including cottonseed, sunflowerseed, safflower seed, rapeseed, leafy greens such as, e.g., lettuce,spinach, kale, collard greens, turnip greens, chard, mustard greens,dandelion greens, broccoli, cabbage, green matter not ordinarilyconsumed by humans, including biomass crops, including switchgrass,miscanthus, sorghum, other grasses, alfalfa, corn stover, green matterordinarily discarded from harvested plants, sugar cane leaves, leaves oftrees, root crops such as cassava, sweet potato, potato, carrots, beets,turnips, plants from the legume family, such as, e.g., clover, peas suchas cowpeas, english peas, yellow peas, green peas, beans such as, e.g.,soybeans, fava beans, lima beans, kidney beans, garbanzo beans, mungbeans, pinto beans, lentils, lupins, mesquite, carob, soy, and peanuts,vetch (vicia), stylo (stylosanthes), arachis, indigofera, acacia,leucaena, cyamopsis, and sesbania. One of skill in the art willunderstand that proteins that can be isolated from any organism in theplant kingdom may be used in the present invention.

Proteins that are abundant in plants can be isolated in large quantitiesfrom one or more source plants and thus are an economical choice for usein any of the muscle, fat, connective tissue replicas, or meatsubstitute products. Accordingly, in some embodiments, the one or moreisolated proteins comprises an abundant protein found in high levels ina plant and capable of being isolated and purified in large quantities.In some embodiments, the abundant protein comprises about 0.5%, 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70% of the total protein content of the source plant. Insome embodiments, the abundant protein comprises about 0.5-10%, about5-40%, about 10-50%, about 20-60%, or about 30-70% of the total proteincontent of the source plant. In some embodiments, the abundant proteincomprises about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50% of the total weight of the dry matter ofthe source plant. In some embodiments, the abundant protein comprisesabout 0.5-5%, about 1-10%, about 5-20%, about 10-30%, about 15-40%,about 20-50% of the total weight of the dry matter of the source plant.

In particular embodiments, the one or more isolated proteins comprisesan abundant protein that is found in high levels in the leaves ofplants. In some embodiments, the abundant protein comprises about 0.5%,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% of the total protein content ofthe leaves of the source plant. In some embodiments, the abundantprotein comprises about 0.5-10%, about 5%-40%, about 10%-60%, about20%-60%, or about 30-70% of the total protein content of the leaves ofthe source plant. In particular embodiments, the one or more isolatedproteins comprise ribulose-1,5-bisphosphate carboxylase oxygenase(rubisco activase). Rubisco is a particularly useful protein for meatreplicas because of its high solubility and an amino acid compositionwith close to the optimum proportions of essential amino acids for humannutrition. In particular embodiments, the one or more isolated proteinscomprise ribulose-1 5-bisphosphate carboxylase oxygenase activase(rubisco activase). In particular embodiments, the one or more isolatedproteins comprise a vegetative storage protein (VSP).

In some embodiments, the one or more isolated proteins include anabundant protein that is found in high levels in the seeds of plants. Insome embodiments, the abundant protein comprises about 0.5%, 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% or more of the total proteincontent of the seeds of the source plant. In some embodiments, theabundant protein comprises about 0.5-10%, about 5%-40%, about 10%-60%,about 20%-60%, or about 30-70% or >70% of the total protein content ofthe seeds of the source plant. Non-limiting examples of proteins foundin high levels in the seeds of plants are seed storage proteins, e.g.,albumins, glycinins, conglycinins, globulins, vicilins, conalbumin,gliadin, glutelin, gluten, glutenin, hordein, prolamin, phaseolin(protein), proteinoplast, secalin, triticeae gluten, zein, any seedstorage protein, oleosins, caloleosins, steroleosins or other oil bodyproteins

In some embodiments, the one or more isolated proteins include proteinsthat interact with lipids and help stabilize lipids in a structure.Without wishing to be bound by a particular theory, such proteins mayimprove the integration of lipids and/or fat replicas with othercomponents of the meat substitute product, resulting in improvedmouthfeel and texture of the final product. A non-limiting example of alipid-interacting plant protein is the oleosin family of proteins.Oleosins are lipid-interacting proteins that are found in oil bodies ofplants. Other non-limiting examples of plant proteins that can stabilizeemulsions include seed storage proteins from Great Northern Beans,albumins from peas, globulins from peas, 8S globulins from Moong bean,8S globulins from Kidney bean.

Muscle Replicas

A large number of meat products comprise a high proportion of skeletalmuscle. Accordingly, the present invention provides a compositionderived from non-animal sources which replicates or approximates keyfeatures of animal skeletal muscle. In another aspect, the presentinvention provides a meat substitute product that comprises acomposition derived from non-animal sources which replicates orapproximates animal skeletal muscle. Such a composition will be labeledherein as “muscle replica”. In some embodiments, the muscle replicaand/or meat substitute product comprising the muscle replica arepartially derived from animal sources. In some embodiments, the musclereplica and/or meat substitute product comprising the muscle replica areentirely derived from non-animal sources.

Many meat products comprise a high proportion of striated skeletalmuscle in which individual muscle fibers are organized mainly in anisotropic fashion. Accordingly, in some embodiments the muscle replicacomprises fibers that are to some extent organized isotropically. Insome embodiments the fibers comprise a protein component. In someembodiments, the fibers comprise about 1%, about 2%, about 5%, about10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%,about 70%, about 80%, about 90%, about 95%, about 99% or more of aprotein component.

In some embodiments, the protein component comprises one or moreisolated, purified proteins. For example the one or more isolated,purified protein can comprise the 8S globulin from Moong bean seeds, orthe albumin or globulin fraction of pea seeds. These proteins provideexamples of proteins with favorable properties for constructing meatreplicas because of their ability to form gels with textures similar toanimal muscle or fat tissue. Examples and embodiments of the one or moreisolated, purified proteins are described herein. The list of potentialcandidates here is essentially open and may include Rubisco, any majorseed storage proteins, proteins isolated from fungi, bacteria, archaea,viruses, or genetically engineered microorganisms, or synthesized invitro. The proteins may be artificially designed to emulate physicalproperties of animal muscle tissue. The proteins may be artificiallydesigned to emulate physical properties of animal muscle tissue. In someembodiments, one or more isolated, purified proteins accounts for about0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 99% or more of the protein component by weight.

Skeletal muscles of animals such as beef cattle typically containsubstantial quantities of glycogen, which can comprise on the order of1% of the mass of the muscle tissue at the time of slaughter. Afterslaughter, a fraction of this glycogen continues to be metabolizedyielding products including lactic acid, which contributes to loweringthe pH of the muscle tissue, a desirable quality in meat. Glycogen is abranched polymer of glucose linked together by alpha (1->4) glycosidicbonds in linear chains, with branch points comprising alpha (1->6)glycosidic bonds. Starches from plants, particularly amylopectins arealso branched polymers of glucose linked together by alpha (1->4)glycosidic bonds in linear chains, with branch points comprising alpha(1->6) glycosidic bonds and can therefore be used as an analog ofglycogen in constructing meat replicas. Thus in some embodiments, themuscle or meat replica includes a starch or pectin.

Additional components of animal muscle tissue include sodium, potassium,calcium, magnesium, other metal ions, lactic acid, other organic acids,free amino acids, peptides, nucleotides and sulfur compounds. Thus insome embodiments, the muscle replica can include sodium, potassium,calcium, magnesium, other metal ions, lactic acid, other organic acids,free amino acids, peptides, nucleotides and sulfur compounds. In someembodiments the concentration of sodium, potassium, calcium, magnesium,other metal ions, lactic acid, other organic acids, free amino acids,peptides, nucleotides and/or sulfur compounds in the muscle replica orconsumable are within 10% of the concentrations found in a muscle ormeat being replicated.

In another aspect, the invention provides methods for making a musclereplica. In some embodiments, the composition is formed into asymmetricfibers prior to incorporation into the consumable. In some embodimentsthese fibers replicate muscle fibers. In some embodiments the fibers arespun fibers. In other embodiments the fibers are extruded fibers.Accordingly, the present invention provides for methods for producingasymmetric or spun protein fibers. In some embodiments, the fibers areformed by extrusion of the protein component through an extruder.Methods of extrusion are well known in the art, and are described inU.S. Pat. No. 6,379,738, U.S. Pat. No. 3,693,533, US20120093994, whichare herein incorporated by reference.

In some embodiments extrusion can be conducted using an MPF19 twin-screwextruder (APV Baker, Grand Rapids, Mich.) with a cooling die. Thecooling die can cool the extrudate prior to return of the extrudate toatmospheric pressure, thus substantially inhibiting expansion or puffingof the final product. In the MPF19 apparatus, dry feed and liquid can beadded separately and mixed in the barrel. Extrusion parameters can be,for example: screw speed of 200 rpm, product temperature at the die of150 C., feed rate of 23 g/min, and water-flow rate of 11 g/min. Producttemperature can be measured during extrusion by a thermocouple at theend of the extrusion barrel. Observations can be made on color, opacity,structure, and texture for each collected sample. Collected samples canbe optionally dried at room temperature overnight, then ground to a finepowder (<60 mesh) using a Braun food grinder. The pH of samples can bemeasured in duplicate using 10% (w/v) slurries of powdered sample indistilled water.

Fat Replica

Animal fat is important for the experience of eating cooked meat.Accordingly, the present invention provides a composition derived fromnon-animal sources which recapitulates key features of animal fat. Inanother aspect, the present invention provides a meat substitute productthat comprises a composition derived from non-animal sources whichrecapitulates animal fat. Such a composition will be labeled herein as a“fat replica”. In some embodiments, the fat replica and/or meatsubstitute product comprising the fat replica are partially derived fromanimal sources.

In some embodiments the meat substitute product has a fat component. Insome embodiments the fat content of the consumable is 1%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% fat. In some embodiments,the fat replica comprises a gel with droplets of fat suspended therein.In some embodiments, the gel is a soft, elastic gel comprising proteinsand optionally carbohydrates. In particular embodiments, the proteinsused in the gel are plant or microbial proteins. In some embodiments,the proteins used in the fat replica might include Rubisco, any majorseed storage proteins, proteins isolated from fungi, bacteria, archaea,viruses, or genetically engineered microorganisms, or synthesized invitro. The proteins may be artificially designed to emulate physicalproperties of animal fat. The proteins may be artificially designed toemulate physical properties of animal fat.

The fat droplets used in some embodiments of the present invention canbe from a variety of sources. In some embodiments, the sources arenon-animal sources. In particular embodiments, the sources are plantsources. Non-limiting examples of oils include corn oil, olive oil, soyoil, peanut oil, walnut oil, almond oil, sesame oil, cottonseed oil,rapeseed oil, canola oil, safflower oil, sunflower oil, flax seed oil,algal oil, palm oil, palm kernel oil, coconut oil, babassu oil, sheabutter, mango butter, cocoa butter, wheat germ oil, rice bran oil, oilsproduced by bacteria, algae, archaea or fungi or genetically engineeredbacteria, algae, archaea or fungi, triglycerides, monoglycerides,diglycerides, sphingosides, glycolipids, lecithin, lysolecithin,phophatidic acids, lysophosphatidic acids, oleic acid, palmitoleic acid,palmitic acid, myristic acid, lauric acid, myristoleic acid, caproicacid, capric acid, caprylic acid, pelargonic acid, undecanoic acid,linoleic acid, 20:1 eicosanoic acid, arachidonic acid, eicosapentanoicacid, docosohexanoic acid, 18:2 conjugated linoleic acid, conjugatedoleic acid, or esters of: oleic acid, palmitoleic acid, palmitic acid,myristic acid, lauric acid, myristoleic acid, caproic acid, capric acid,caprylic acid, pelargonic acid, undecanoic acid, linoleic acid, 20:1eicosanoic acid, arachidonic acid, eicosapentanoic acid, docosohexanoicacid, 18:2 conjugated linoleic acid, or conjugated oleic acid, orglycerol esters of oleic acid, palmitoleic acid, palmitic acid, myristicacid, lauric acid, myristoleic acid, caproic acid, capric acid, caprylicacid, pelargonic acid, undecanoic acid, linoleic acid, 20:1 eicosanoicacid, arachidonic acid, eicosapentanoic acid, docosohexanoic acid, 18:2conjugated linoleic acid, or conjugated oleic acid, or triglyceridederivatives of oleic acid, palmitoleic acid, palmitic acid, myristicacid, lauric acid, myristoleic acid, caproic acid, capric acid, caprylicacid, pelargonic acid, undecanoic acid, linoleic acid, 20:1 eicosanoicacid, arachidonic acid, eicosapentanoic acid, docosohexanoic acid, 18:2conjugated linoleic acid, or conjugated oleic acid.

In some embodiments, fat droplets are derived from pulp or seed oil. Inother embodiments, the source may be yeast or mold. For instance, in oneembodiment the fat droplets comprise triglycerides derived fromMortierella isabellina.

In some embodiments plant oils are modified to resemble animal fats. Theplant oils can be modified with flavoring or other agents torecapitulate the taste and smell of meat during and after cooking.Accordingly, some aspects of the invention involve methods for testingthe qualitative similarity between the cooking properties of animal fatand the cooking properties of plant oils in the consumable.

In some embodiments, the fat replica comprises a protein componentcomprising one or more isolated, purified proteins. The purifiedproteins contribute to the taste and texture of the meat replica. Insome embodiments purified proteins can stabilize emulsified fats. Insome embodiments the purified proteins can form gels upon denaturationor enzymatic crosslinking, which replicate the appearance and texture ofanimal fat. Examples and embodiments of the one or more isolated,purified proteins are described herein. In particular embodiments, theone or more isolated proteins comprise a protein isolated from thelegume family of plants. Non-limiting examples of legume plants aredescribed herein, although variations with other legumes are possible.In some embodiments, the legume plant is a pea plant. In someembodiments the isolated purified proteins stabilize emulsions. In someembodiments the isolated purified proteins form gels upon crosslinkingor enzymatic crosslinking. In some embodiments, the isolated, purifiedproteins comprise seed storage proteins. In some embodiments, theisolated, purified proteins comprise albumin. In some embodiments, theisolated, purified proteins comprise globulin. In a particularembodiment, the isolated, purified protein is a purified pea albuminprotein. In another particular embodiment, the isolated, purifiedprotein is a purified pea globulin protein. In another particularembodiment the isolate purified protein is a Moong bean 8S globulin. Inanother particular embodiment, the isolated, purified protein is anoleosin. In another particular embodiment, the isolated, purifiedprotein is a caloleosin. In another particular embodiment, the isolated,purified protein is Rubisco. In some embodiments, the protein componentcomprises about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more of the fatreplica by dry weight or total weight. In some embodiments, the proteincomponent comprises about 0.1-5%, about 0.5-10%, about 1-20%, about5-30%, about 10-50%, about 20-70%, or about 30-90% or more of the fatreplica by dry weight or total weight. In some embodiments, the proteincomponent comprises a solution containing one or more isolated, purifiedproteins.

In some embodiments, the fat replica comprises cross-linking enzymesthat catalyze reactions leading to covalent crosslinks between proteins.Cross-linking enzymes can be used to create or stabilize the desiredstructure and texture of the adipose tissue replica, to mimic thedesired texture of an equivalent desired animal fat. Non-limitingexamples of cross-linking enzymes include, e.g., transglutaminase, lysyloxidases, or other amine oxidases (e.g. Pichia pastoris lysyl oxidase).In some embodiments, the cross-linking enzymes are isolated and purifiedfrom a non-animal source, examples and embodiments of which aredescribed herein. In some embodiments, the fat replica comprises atleast 0.0001%, or at least 0.001%, or at least 0.01%, or at least 0.1%,or at least 1% (wt/vol) of a cross-linking enzyme. In particularembodiments, the cross-linking enzyme is transglutaminase.

In another aspect, the invention provides methods for making a fatreplica. In some embodiments, the fat droplets are suspended in a gel.In some embodiments the present invention provides for methods forproducing droplets of fat suspended in the gel. The fat can isolated andhomogenized. For example an organic solvent mixture can be used to helpmix a lipid. The solvent can then be removed. At this point the lipidcan be frozen, lyophilized, or stored. So in some aspects the inventionprovides for a method for isolating and storing a lipid which has beenselected to have characteristics similar to animal fat. The lipid filmor cake can then be hydrated. The hydration can utilize agitation ortemperature changes. The hydration can occur in a precursor solution toa gel. After hydration the lipid suspension can be sonicated or extrudedto further alter the properties of the lipid in the solution.

In some embodiments, the fat replica is assembled to approximate theorganization adipose tissue in meat. In some embodiments some or all ofthe components of the fat replica are suspended in a gel. In variousembodiments the gel can be a proteinaceous gel, a hydrogel, anorganogel, or a a xerogel. In some embodiments, the gel can be thickenedto a desired consistency using an agent based on polysaccharides orproteins. For example fecula, arrowroot, cornstarch, katakuri starch,potato starch, sago, tapioca, alginin, guar gum, locust bean gum,xanthan gum, collagen, egg whites, furcellaran, gelatin, agar,carrageenan, cellulose, methylcellulose, hydroxymethylcellulose, acadiagum, konjac, starch, pectin, amylopectin or proteins derived fromlegumes, grains, nuts, other seeds, leaves, algae, bacteria, of fungican be used alone or in combination to thicken the gel, forming anarchitecture or structure for the consumable.

In particular embodiments, the fat replica is an emulsion comprising asolution of one or more proteins and one or more fats suspended thereinas droplets. In some embodiments, the emulsion is stabilized by one ormore cross-linking enzymes into a gel. In more particular embodiments,the one or more proteins in solution are isolated, purified proteins. Inyet more particular embodiments, the isolated, purified proteinscomprise a purified pea albumin enriched fraction. In other moreparticular embodiments, the isolated, purified proteins comprise apurified pea globulin enriched fraction. In other more particularembodiments, the isolated, purified proteins comprise a purified Moongbean 8S globulin enriched fraction. In yet more particular embodiments,the isolated, purified proteins comprise a Rubisco enriched fraction. Inother particular embodiments, the one or more fats are derived fromplant-based oils. In more particular embodiments, the one or more fatsare derived from one or more of: corn oil, olive oil, soy oil, peanutoil, walnut oil, almond oil, sesame oil, cottonseed oil, rapeseed oil,canola oil, safflower oil, sunflower oil, flax seed oil, algal oil, palmoil, palm kernel oil, coconut oil, babassu oil, shea butter, mangobutter, cocoa butter, wheat germ oil, rice bran oil, oils produced bybacteria, algae, archaea or fungi or genetically engineered bacteria,algae, archaea or fungi, triglycerides, monoglycerides, diglycerides,sphingosides, glycolipids, lecithin, lysolecithin, phophatidic acids,lysophosphatidic acids, oleic acid, palmitoleic acid, palmitic acid,myristic acid, lauric acid, myristoleic acid, caproic acid, capric acid,caprylic acid, pelargonic acid, undecanoic acid, linoleic acid, 20:1eicosanoic acid, arachidonic acid, eicosapentanoic acid, docosohexanoicacid, 18:2 conjugated linoleic acid, conjugated oleic acid, or estersof: oleic acid, palmitoleic acid, palmitic acid, myristic acid, lauricacid, myristoleic acid, caproic acid, capric acid, caprylic acid,pelargonic acid, undecanoic acid, linoleic acid, 20:1 eicosanoic acid,arachidonic acid, eicosapentanoic acid, docosohexanoic acid, 18:2conjugated linoleic acid, or conjugated oleic acid, or glycerol estersof oleic acid, palmitoleic acid, palmitic acid, myristic acid, lauricacid, myristoleic acid, caproic acid, capric acid, caprylic acid,pelargonic acid, undecanoic acid, linoleic acid, 20:1 eicosanoic acid,arachidonic acid, eicosapentanoic acid, docosohexanoic acid, 18:2conjugated linoleic acid, or conjugated oleic acid, or triglyceridederivatives of oleic acid, palmitoleic acid, palmitic acid, myristicacid, lauric acid, myristoleic acid, caproic acid, capric acid, caprylicacid, pelargonic acid, undecanoic acid, linoleic acid, 20:1 eicosanoicacid, arachidonic acid, eicosapentanoic acid, docosohexanoic acid, 18:2conjugated linoleic acid, or conjugated oleic acid. In yet even moreparticular embodiments, the one or more fats is a rice bran oil. Inanother particular embodiment, the one or more fats is a canola oil. Inother particular embodiments, the cross-linking enzyme istransglutaminase, lysyl oxidase, or other amine oxidase. In yet evenmore particular embodiments, the cross-linking enzyme istransglutaminase. In particular embodiments, the fat replica is a highfat emulsion comprising a protein solution of purified pea albuminemulsified with 40-80% rice bran oil, stabilized with 0.5-5% (wt/vol)transglutaminase into a gel. In particular embodiments, the fat replicais a high fat emulsion comprising a protein solution ofpartially-purified moong bean 8S globulin emulsified with 40-80% ricebran oil, stabilized with 0.5-5% (wt/vol) transglutaminase into a gel.In particular embodiments, the fat replica is a high fat emulsioncomprising a protein solution of partially-purified moong bean 8Sglobulin emulsified with 40-80% canola oil, stabilized with 0.5-5%(wt/vol) transglutaminase into a gel. In particular embodiments, the fatreplica is a high fat emulsion comprising a protein solution of purifiedpea albumin emulsified with 40-80% rice bran oil, stabilized with0.0001-1% (wt/vol) transglutaminase into a gel. In particularembodiments, the fat replica is a high fat emulsion comprising a proteinsolution of partially-purified moong bean 8S globulin emulsified with40-80% rice bran oil, stabilized with 0.0001-1% (wt/vol)transglutaminase into a gel. In particular embodiments, the fat replicais a high fat emulsion comprising a protein solution ofpartially-purified moong bean 8S globulin emulsified with 40-80% canolaoil, stabilized with 0.0001-1% (wt/vol) transglutaminase into a gel.

Connective Tissue Replica

Animal connective tissue provides key textural features that are animportant component of the experience of eating meat. Accordingly, thepresent invention provides a composition derived from non-animal sourceswhich recapitulates key features of animal connective tissue. In anotheraspect, the present invention provides a meat substitute product thatcomprises a composition derived from non-animal sources whichrecapitulates important textural and visual features of animalconnective tissue. Such a composition will be labeled herein as“connective tissue replica”. In some embodiments, the connective tissuereplica and/or meat substitute product comprising the connective tissuereplica are partially derived from animal sources.

Animal connective tissue can generally be divided into fascia-type andcartilage-type tissue. Fascia-type tissue is highly fibrous, resistantagainst extension (has high elastic modulus), and has a high proteincontent, a moderate water content (ca. 50%), and low-to-none fat andpolysaccharide content. Accordingly, the present invention provides aconnective tissue replica that recapitulates key features of fascia typetissue. In some embodiments, the connective tissue replica comprisesabout 50% protein by total weight, about 50% by liquid weight, and has alow fat and polysaccharide component.

The protein content of most fascia-type connective tissue is comprisedmainly of collagen. Collagen is characterized by a high fraction ofproline and alanine, and also is assembled into characteristic elongatedfibrils or rod-like, flexible structures. Prolamins are one family ofproteins found in non-animal sources, such as plant sources. Prolaminsare highly abundant in plants and are similar in amino acid compositionto collagen. Among proteins we tested for this purpose, prolamins wereparticularly favorable because of their low cost and their ability toreadily form fibers or sheets when spun or extruded. Non-limitingexamples of prolamin family proteins include, e.g., zein (found incorn), these include hordein from barley, gliadin from wheat, secalin,extensins from rye, kafirin from sorghum, avenin from oats. Infascia-type connective tissue, the prolamin family of proteins,individually or combinations thereof, demonstrates suitability for theprotein component because they are highly abundant, similar in globalamino acid composition to collagen (high fraction of proline andalanine), and amenable to processing into films and fibers. In additionto zein (found in corn), these include hordein from barley, gliadin fromwheat, secalin, extensins from rye, kafirin from sorghum, avenin fromoats. Other proteins may be necessary to supplement prolamins in orderto achieve targets specifications for physicochemical and nutritionalproperties. The list of potential candidates here is essentially openand may include Rubisco, any major seed storage proteins, proteinsisolated from fungi, bacteria, archaea, viruses, or geneticallyengineered microorganisms, or synthesized in vitro. The proteins may beartificially designed to emulate physical properties of animalconnective tissue. Animal-derived or recombinant collagen, extensins(hydroxyproline-rich glycoproteins abundant in cell walls e.g.Arabidopsis thaliana, monomers of which are “collagen-like” rod-likeflexible molecules). The proteins may be artificially designed toemulate physical properties of animal connective tissue.

Methods for forming fascia-type connective tissue will be as thosepracticed in the art with a bias towards methods producing fibrous orfibrous-like structures by biological, chemical, or physical means,individually or in combination, serially or in parallel, before finalforming. These methods may include extrusion or spinning.

Cartilage-type tissue is macroscopically homogenous, resistant againstcompression, has higher water content (up to 80%), lower protein(collagen) content, and higher polysaccharide (proteoglycans) contents(ca. 10% each).

Compositionally, cartilage-type connective tissue will be very similarto fascia-type tissue with the relative ratios of each adjusted to moreclosely mimic ‘meat’ connective tissue.

Methods for forming cartilage-type connective tissue will be similar tothose for fascia-type connective tissue, but with a bias towards methodsproducing isotropically homogenous structures.

The fat can be suspended in a gel. In some embodiments the presentinvention provides for methods for producing droplets of fat suspendedin the proteinaceous gel. The fat can be isolated from plant tissues andemulsified. The emulsification can utilize high-speed blending,homogenization, agitation or temperature changes. The lipid suspensioncan be sonicated or extruded to further alter the properties of thelipid in the solution. At this point, in some embodiments othercomponents of the consumable are added to the solution followed by agelling agent. In some embodiments crosslinking agents (e.g.transglutaminase or lysyl oxidase) are added to bind the components ofthe consumable. In other embodiments the gelling agent is added and thelipid/gel suspension is later combined with additional components of theconsumable. In fascia-type connective tissue, the prolamin family ofproteins, individually or combinations thereof, demonstrates suitabilityfor the protein component because they are highly abundant, similar inglobal amino acid composition to collagen (high fraction of proline andalanine), and amenable to processing into films. In addition to zein(found in corn), these include hordein from barley, gliadin from wheat,secalin, extensions from rye, kafirin from sorghum, avenin from oats.Other proteins may be necessary to supplement prolamins in order toachieve targets specifications for physicochemical and nutritionalproperties. The list of potential candidates here is essentially openand may include any major seed storage proteins, animal-derived orrecombinant collagen, extensins (hydroxyproline-rich glycoproteinsabundant in cell walls e.g. Arabidopsis thaliana, monomers of which are“collagen-like” rod-like flexible molecules).

In some embodiments some or all of the components of the consumable aresuspended in a gel. In various embodiments the gel can be a hydrogel, anorganogel, or a xerogel, The gel can be made thick using an agent basedon polysaccharides or proteins. For example fecula, arrowroot,cornstarch, katakuri starch, potato starch, sago, tapioca, alginin, guargum, locust bean gum, xanthan gum, collagen, egg whites, furcellaran,gelatin, agar, carrageenan, cellulose, methylcellulose,hydroxymethylcellulose, acadia gum, konjac, starch, pectin, amylopectinor proteins derived from legumes, grains, nuts, other seeds, leaves,algae, bacteria, of fungi can be used alone or in combination to thickenthe gel, forming an architecture or structure for the consumable.Enzymes that catalyze reactions leading to covalent crosslinks betweenproteins can also be used alone or in combination to form anarchitecture or structure for the consumable. For exampletransclutaminase, lysyl oxidases, or other amine oxidases (e.g. Pichiapastoris lysyl oxidase (PPLO)) can be used alone or in combination toform an architecture or structure for the consumable. In someembodiments multiple gels with different components are combined to formthe consumable. For example a gel containing a plant-based protein canbe associated with a gel containing a plant-based fat. In someembodiments fibers or stings of proteins are oriented parallel to oneanother and then held in place by the application of a gel containingplant based fats.

The compositions of the invention can be puffed or expanded by heating,such as frying, baking, microwave heating, heating in a forced airsystem, heating in an air tunnel, and the like, according to methodswell known in the art.

In some embodiments multiple gels with different components are combinedto form the consumable. For example a gel containing a plant-basedprotein can be associated with a gel containing a plant-based fat. Insome embodiments fibers or strings of proteins are oriented parallel toone another and then held in place by the application of a gelcontaining plant based fats.

In some embodiments the meat replica contains no animal products, lessthan 1% wheat gluten, no methylcellulose, no carrageenan, no caramelcolor and no Konjac flour, no gum Arabic, and no acacia gum.

In some embodiments the meat replica contains no animal products, nowheat gluten, no methylcellulose, no carrageenan, no caramel color andno Konjac flour, no gum Arabic, and no acacia gum.

In some embodiments the meat replica contains no animal products, no soyprotein isolate, no wheat gluten, no methylcellulose, no carrageenan, nocaramel color and no Konjac flour, no gum Arabic, and no acacia gum.

In some embodiments the meat replica contains no animal products, no soyprotein concentrate, no wheat gluten, no methylcellulose, nocarrageenan, no caramel color and no Konjac flour, no gum Arabic, and noacacia gum.

In some embodiments the meat replica contains no animal products, no soyprotein, no wheat gluten, no methylcellulose, no carrageenan, no caramelcolor and no Konjac flour, no gum Arabic, and no acacia gum.

In some embodiments the meat replica contains no animal products, notofu, no wheat gluten, no methylcellulose, no carrageenan, no caramelcolor and no Konjac flour, no gum Arabic, and no acacia gum.

In some embodiments the meat replica contains no animal products, notofu, and no wheat gluten.

In some embodiments the meat replica contains no animal products, no soyprotein, and no wheat gluten.

In some embodiments the meat replica contains no methylcellulose, nocarrageenan, no caramel color, no Konjac flour, no gum Arabic, and noacacia gum.

In some embodiments the meat replica contains no animal products andless than 5% carbohydrates.

In some embodiments the meat replica contains no animal products, no soyprotein, no wheat gluten, no methylcellulose, no carrageenan, no caramelcolor and no Konjac flour, no gum Arabic, and no acacia gum and lessthan 5% carbohydrates.

In some embodiments the meat replica contains no animal products, andless than 1% cellulose.

In some embodiments the meat replica contains no animal products, andless than 5% insoluble carbohydrates.

In some embodiments the meat replica contains no animal products, no soyprotein, and less than 1% cellulose.

In some embodiments the meat replica contains no animal products, no soyprotein, and less than 5% insoluble carbohydrates.

In some embodiments the meat replica contains no animal products, nowheat gluten, and less than 1% cellulose.

In some embodiments the meat replica contains no animal products, nowheat gluten, and less than 5% insoluble carbohydrates.

The percentage of different components may also be controlled. Forexample non-animal-based substitutes for muscle, fat tissue, connectivetissue, and blood components can be combined in different ratios andphysical organizations to best approximate the look and feel of meat.The various can also components can be arranged to insure consistencybetween bites of the consumable. The components can be arranged toinsure that no waste is generated from the consumable. For example,while a traditional cut of meat may have portions that are not typicallyeaten, a meat replicate can improve upon meat by not including theseinedible portions. Such an improvement allows for all of the productmade or shipped to be consumed, which cuts down on waste and shippingcosts. Alternatively, a meat replica may include inedible portions tomimic the experience of meat consumption. Such portions can includebone, cartilage, connective tissue, or other materials commonly referredto as gristle, or materials included simulating these components. Insome embodiments the consumable may contain simulated inedible portionsof meat products which are designed to serve secondary functions. Forexample a simulated bone can be designed to disperse heat duringcooking, making the cooking of the consumable faster or more uniformthan meat. In other embodiments a simulated bone may also serve to keepthe consumable at a constant temperature during shipping. In otherembodiments, the simulated inedible portions may be biodegradable.

In some embodiments the meat substitute compositions contains no animalprotein, comprising between 10-30% protein, between 5-80% water, between5-70% fat, comprising one or more isolated purified proteins. Inparticular embodiments, the meat substitute compositions comprisetransglutaminase.

In some embodiments the consumable contains components to replicate thecomponents of meat. The main component of meat is typically skeletalmuscle. Skeletal muscle typically consists of roughly 75 percent water,19 percent protein, 2.5 percent intramuscular fat, 1.2 percentcarbohydrates and 2.3 percent other soluble non-protein substances.These include organic acids, sulfur compounds, nitrogenous compounds,such as amino acids and nucleotides, and inorganic substances such asminerals. Accordingly, some embodiments of the present invention providefor replicating approximations of this composition for the consumable. For example, in some embodiments the consumable is a plant-based meatreplica can comprise roughly 75% water, 19% protein, 2.5% fat, 1.2%carbohydrates; and 2.3 percent other soluble non-protein substances. Insome embodiments the consumable is a plant-based meat replica comprisingbetween 60-90% water, 10-30% protein, 1-20% fat, 0.1-5% carbohydrates;and 1-10 percent other soluble non-protein substances. In someembodiments the consumable is a plant-based meat replica comprisingbetween 60-90% water, 5-10% protein, 1-20% fat, 0.1-5% carbohydrates;and 1-10 percent other soluble non-protein substances. In someembodiments the consumable is a plant-based meat replica comprisingbetween 0-50% water, 5-30% protein, 20-80%% fat, 0.1-5% carbohydrates;and 1-10 percent other soluble non-protein substances. In someembodiments, the replica contains between 0.01% and 5% by weight of aheme protein. In some embodiments, the replica contains between 0.01%and 5% by weight of leghemoglobin. Some meat also contains myoglobin, aheme protein, which accounts for most of the red color and iron contentof some meat. In some embodiments, the replica contains between 0.01%and 5% by weight of a heme protein. In some embodiments, the replicacontains between 0.01% and 5% by weight of leghemoglobin. It isunderstood that these percentages can vary in meat and the meat replicascan be produced to approximate the natural variation in meat.Additionally, in some instances, the present invention provides forimproved meat replicas, which comprise these components in typicallyunnatural percentages. For example a meat replica can be produced with ahigher than typical average fat content. The percentages of thesecomponents may also be altered to increase other desirable properties.

In some instances a meat replica is designed so that, when cooked, thepercentages of components are similar to cooked meat. So, in someembodiments, the uncooked consumable has different percentages ofcomponents than uncooked meat, but when cooked the consumable is similarto cooked meat. For example, a meat replica may be made with a higherthan typical water content for raw meat, but when cooked in a microwavethe resulting product has percentages of components similar to meatcooked over a fire.

In some embodiments the consumable is a meat replica with a lower thattypical water content for meat. In some embodiments the inventionsprovides for methods for hydrating a meat replica to cause the meatreplica to have a content similar to meat. For example a meat replicawith a water content that would be low for meat, for example 1%, 10%,20%, 30%, 40% or 50% water, is hydrated to roughly 75% water. Oncehydrated, in some embodiments, the meat replica is then cooked for humanconsumption.

The consumable can have a protein component. In some embodiments theprotein content of the consumable is 10%, 20%, 30%, or 40%. In someembodiments the protein content of the consumable is similar to meat. Insome embodiments the protein content in the consumable is greater thanthat of meat. In some embodiments the consumable has less protein thanmeat.

The protein in the consumable can come from a variety or combination ofsources. Non-animal sources can provide some or all of the protein inthe consumable. Non-animal sources can include vegetables, fruits, nuts,grains, algae, bacteria, or fungi. The protein can be isolated orconcentrated from one or more of these sources. In some embodiments theconsumable is a meat replica comprising protein only obtained fromnon-animal sources.

In some embodiments protein is formed into asymmetric fibers forincorporation into the consumable. In some embodiments these fibersreplicate muscle fibers. In some embodiments the protein are spunfibers. Accordingly, the present invention provides for methods forproducing asymmetric or spun protein fibers. In some embodiments theconsumable contains a protein or proteins that have all of the aminoacids found in proteins that are essential for human nutrition. In someembodiments the proteins added to the consumable are supplemented withamino acids.

Indicators of Cooking Meat

The release of odorants upon cooking is an important aspect of meatconsumption. In some embodiments, the consumable is a meat replicaentirely composed of non-animal products that when cooked generates anaroma recognizable by humans as typical of cooking beef. In someembodiments, the consumable when cooked generates an aroma recognizableby humans as typical of cooking pork. In some embodiments, theconsumable is a meat replica entirely composed of non-animal productsthat when cooked generates an aroma recognizable by humans as typical ofcooking bacon. In some embodiments, the consumable is a meat replicaentirely composed of non-animal products that when cooked generates anaroma recognizable by humans as typical of cooking chicken. In someembodiments, the consumable is a meat replica entirely composed ofnon-animal products that when cooked generates an aroma recognizable byhumans as typical of cooking lamb. In some embodiments, the consumableis a meat replica entirely composed of non-animal products that whencooked generates an aroma recognizable by humans as typical of cookingfish. In some embodiments, the consumable is a meat replica entirelycomposed of non-animal products that when cooked generates an aromarecognizable by humans as typical of cooking turkey. In some embodimentsthe consumable is a meat replica principally or entirely composed ofingredients derived from non-animal sources, with an odorant that isreleased upon cooking. In some embodiments the consumable is a meatreplica principally or entirely composed of ingredients derived fromnon-animal sources, with an odorant that is produced by chemicalreactions that take place upon cooking. In some embodiments theconsumable is a meat replica principally or entirely composed ofingredients derived from non-animal sources, containing mixtures ofproteins, peptides, amino acids, nucleotides, sugars and polysaccharidesand fats in combinations and spatial arrangements that enable thesecompounds to undergo chemical reactions during cooking to produceodorants and flavor-producing compounds. In some embodiments theconsumable is a meat replica principally or entirely composed ofingredients derived from non-animal sources, with a volatile or labileodorant that is released upon cooking. In some embodiments theconsumable is a method for preparing a meat replica where meat replicasprincipally or entirely composed of ingredients derived from non-animalsources are heated to release a volatile or labile odorant.

Odorants released during cooking of meat are generated by reactions thatcan involve as reactants fats, protein, amino acids, peptides,nucleotides, organic acids, sulfur compounds, sugars and othercarbohydrates. In some embodiments the odorants that combine during thecooking of meat are identified and located near one another in theconsumable, such that upon cooking of the consumable the odorantscombine. So, in some embodiments, the characteristic flavor andfragrance components are produced during the cooking process by chemicalreactions involving amino acids, fats and sugars found in plants as wellas meat. So, in some embodiments, the characteristic flavor andfragrance components are mostly produced during the cooking process bychemical reactions involving one or more amino acids, fats, peptides,nucleotides, organic acids, sulfur compounds, sugars and othercarbohydrates found in plants as well as meat.

Some reactions that generate odorants released during cooking of meatcan be catalyzed by iron, in particular the heme iron of myoglobin. Thusin some embodiments, some of the characteristic flavor and fragrancecomponents are produced during the cooking process by chemical reactionscatalyzed by iron. In some embodiments, some of the characteristicflavor and fragrance components are produced during the cooking processby chemical reactions catalyzed by heme. In some embodiments, some ofthe characteristic flavor and fragrance components are produced duringthe cooking process by chemical reactions catalyzed by the heme iron inleghemoglobin. In some embodiments, some of the characteristic flavorand fragrance components are produced during the cooking process bychemical reactions catalyzed by the heme iron in a heme protein.

Evidence that the presence of leghemoglobin contributes favorably toaroma of meat replicas: A muscle replica comprising pea flour, sunfloweroil, and glucose was heated for 10 minutes at 140 C in the presence ofeither reduced leghemoglobin (LHb) or a mixture of iron (Fe3+), sodiumand EDTA (EFS) in sealed containers carrying solid phase microextraction(SPME) fibers. These fibers contain polydimethylsiloxane (PDMS) whichadsorbs volatile compounds for analysis by GC-MS. Analysis of GC-MS datafrom multiple replicas reveal consistent differences between the LHb andEFS samples. Non-limiting examples of compounds found exclusively ormore abundantly in the LHb samples are: 2-octanone, 2-methyl furan,which are often associated with the aroma of cooked meat, and many otherunidentified compounds.

Color Indicators

The color of meat is an important part the experience of cooking andeating meat. For instance, cuts of beef are of a characteristic redcolor in a raw state and gradually transition to a brown color duringcooking. As another example, white meats such as chicken or pork have acharacteristic pink color in their raw state and gradually transition toa white or brownish color during cooking. The amount of the colortransition is used to indicate the cooking progression of beef andtitrate the cooking time and temperature to produce the desired state ofdone-ness. In some aspects, the invention provides a non-meat based meatsubstitute product that provides a visual indicator of cookingprogression. In some embodiments, the visual indicator is a colorindicator that undergoes a color transition during cooking. Inparticular embodiments, the color indicator recapitulates the colortransition of a cut of meat as the meat progresses from a raw to acooked state. In more particular embodiments, the color indicator colorsthe meat substitute product a red color before cooking to indicate a rawstate and causes the meat substitute product to transition to a browncolor during cooking progression. In other particular embodiments, thecolor indicator colors the meat substitute product a pink color beforecooking to indicate a raw state and causes the meat substitute productto transition to a white or brown color during cooking progression.

The main determinant of the nutritional definition of the color of meatis the concentration of iron carrying proteins in the meat. In theskeletal muscle component of meat products, one of the mainiron-carrying proteins is myoglobin. It is estimated that the white meatof chicken has under 0.05%; pork and veal have 0.1-0.3%; young beef has0.4-1.0%; and old beef has 1.5-2.0%. So, in some embodiments, theconsumable is a meat replica which comprises an iron-carrying protein.In some embodiments, the meat replica comprises about 0.05%, about 0.1%,about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%,about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%,about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%,about 2%, or more than about 2% of an iron-carrying protein by dryweight or total weight. In some cases, the iron carrying protein hasbeen isolated and purified from a source. In other cases, the ironcarrying protein has not been isolated and purified. In some cases, thesource of the iron-carrying protein is an animal source, or a non-animalsource such as a plant, fungus, or genetically modified organisms suchas, e.g., bacteria or yeast. In some cases, the iron-carrying protein ismyoglobin. In some embodiments the consumable is a plant based meatreplica that has animal myoglobin added. So, for example a replica ofyoung beef can have about 0.4-1% myoglobin. In some cases, theiron-carrying protein is leghemoglobin. In some embodiments theconsumable is a plant based meat replica that has leghemoglobin added.So, for example a replica of young beef can have about 0.4-1%leghemoglobin. In some cases, the iron-carrying protein is a cytochrome.In some embodiments the consumable is a plant based meat replica thathas a cytochrome added. So, for example a replica of young beef can haveabout 0.4-1% of a cytochrome.

Another example of iron-carrying proteins is hemoglobin, theiron-containing oxygen-binding protein in the red blood cells ofvertebrates. Hemoglobin is similar in color to myoglobin. In someembodiments the invention provides methods of saving and recycling bloodfrom animal farming to supplement the color of a consumable. For exampleblood is saved from a slaughter house, hemoglobin from the blood is usedto enhance the color of a consumable. In some aspects the consumable isa plant-based meat replica containing hemoglobin.

Additional iron containing proteins exist in nature. In some embodimentsthe consumable comprises an iron containing protein that is notmyoglobin. In some embodiments the consumable does not containmyoglobin. In some embodiments the consumable does not containhemoglobin. In some embodiments the consumable is a meat replica thatcomprises an iron containing protein other than myoglobin or hemoglobin.

Examples of iron containing proteins include hemoglobin, myoglobin,neuroglobin, cytoglobin, leghemoglobin, non-symbiotic hemoglobin, Hell'sgate globin I, bacterial hemoglobins, ciliate myoglobins,flavohemoglobins. In various embodiments these iron containing proteinsare added to the consumable to alter the visual characteristics or ironcontent of the consumable. In some embodiments the consumable comprisesa hemoprotein (e.g. hemoglobin, myoglobin, neuroglobin, cytoglobin,leghemoglobin, non-symbiotic hemoglobin, Hell's gate globin I, bacterialhemoglobins, ciliate myoglobins, flavohemoglobins).

Leghemoglobin, similar in structure and physical properties tomyoglobin, is readily available as an unused by-product of commoditylegume crops (e.g., soybean, pea). The leghemoglobin in the roots ofthese crops in the US exceeds the myoglobin content of all the red meatconsumed in the US. In some embodiments the consumable is a meat replicaprincipally or entirely composed of ingredients derived from non-animalsources, including a muscle tissue replica, an adipose tissue replica, aconnective tissue replica, and leghemoglobin. In some embodiments theconsumable is a meat replica principally or entirely composed ofingredients derived from non-animal sources, containing a heme protein.In some embodiments the consumable is a meat replica principally orentirely composed of ingredients derived from non-animal sources,containing a leghemoglobin. In some embodiments the consumable is a meatreplica principally or entirely composed of ingredients derived fromnon-animal sources, containing a member of the globin protein family. Insome embodiments the consumable is a meat replica principally orentirely composed of ingredients derived from non-animal sources, with ahigh iron content from a heme protein. In some embodiments the ironcontent is similar to meat. In some embodiments the consumable has thedistinctive red color of meat, such color provided by leghemoglobin.

Leghemoglobin is, in some embodiments, used as an indicator that theconsumable is finished cooking. So, one embodiment of the invention is amethod for cooking a consumable comprising detecting leghemoglobin whichhas migrated from the interior of the consumable to the surface when theproduct is cooked. Another embodiment of the invention is a method forcooking a consumable comprising detecting the change in color of fromred to brown when the product is cooked.

A heme protein is, in some embodiments, used as an indicator that theconsumable is finished cooking. So, one embodiment of the invention is amethod for cooking a consumable comprising detecting leghemoglobin whichhas migrated from the interior of the consumable to the surface when theproduct is cooked. Another embodiment of the invention is a method forcooking a consumable comprising detecting the change in color of fromred to brown when the product is cooked.

A heme protein from the group of: Hemoglobin, myoglobin, neuroglobin,cytoglobin, leghemoglobin, non-symbiotic hemoglobin, Hell's gate globinI, bacterial hemoglobins, ciliate myoglobins, flavohemoglobins, is, insome embodiments, used as an indicator that the consumable is finishedcooking. So, one embodiment of the invention is a method for cooking aconsumable comprising detecting leghemoglobin which has migrated fromthe interior of the consumable to the surface when the product iscooked. Another embodiment of the invention is a method for cooking aconsumable comprising detecting the change in color of from red to brownwhen the product is cooked.

Food Products Comprising Isolated, Purified Leghemoglobin

In some embodiments leghemoglobin is added to meat to enhance theproperties of meat. For example, a leghemglobin containing solution canbe injected into raw or cooked meat. In another example a leghemoglobinsolution is dripped over meat or a consumable of the invention toenhance appearance. In one embodiment advertising, photography, orvideography of food products such as meat or a meat substitute isenhanced with leghemoglobin.

Sources of Leghemoglobin

In some embodiments the present invention provides methods for obtainingleghemoglobin from plants. Leghemoglobin can be obtained from a varietyof plants. Various legumes species and their varieties, for example,Soybean, Fava bean, Lima bean, Cowpeas, English peas, Yellow peas,Lupine, Kidney bean, Garbanzo beans, Peanut, Alfalfa, Vetch hay, Clover,Lespedeza and Pinto bean, contain nitrogen-fixing root nodules in whichleghemoglobin has a key role in controlling oxygen concentrations (forexample root nodules from a pea plant, FIG. 1). FIG. 2 shows 100 mls ofleghemoglobin solution isolated from 30 grams of pea root nodules.Leghemoglobins from different species are homologs and have similarcolor properties (FIG. 3). In FIG. 3, panel A shows an SDS_PAGE gels oflysed root-nodules of three legume plant species (1) Fava bean (2)English Pea (3) Soybean. Arrows mark respective leghemoglobins. Notethat leghemoglobin is the most abundant soluble protein in each lysate.Panel B shows the similarity of UV-VIS spectral profile ofleghemoglobins from two different plant species (Favabean and Soybean).We purified leghemoglobin from fava bean (green curve) and Soybean (redcurve) root nodules using the protocol described elsewhere in thespecification. UV-VIS spectra of both proteins shows that the heme ironis in the reduced (+2) state. Note that they are almost perfectlysuperimposed, consistent with their visually identical red color. Theheme iron in the respective leghemoglobins was reduced to the +2oxidation state by incubating Fava bean and Soybean leghemoglobin with10 mM sodium hydrosulfite in 20 mM potassium-phosphate pH 7.4, 100 mMsodium chloride buffer. Sodium hydrosulfite was then removed from theleghemoglobin solution using gel-exclusion chromatography. Inset shows azoom-in of UV-VIS spectra in 450 nm to 700 nm region. Some plant speciesexpress several leghemoglobin isoforms (for example soybean has fourleghemoglobin isoforms). Minor variations in precise amino acid sequencecan modify overall charge of the protein at a particular pH and canmodify precise structural conformation of iron containing heme group inleghemoglobin. Differences in structural conformation of heme group ofdifferent leghemoglobins can influence oxidation and reduction rates ofthe heme iron. These differences may contribute to color and flavorgeneration properties of different leghemoglobins.

Leghemoglobin has a virtually identical absorbance spectrum and visualappearance to myoglobin from animal muscle. FIG. 4 shows a comparison ofreduced (heme iron 2+) and oxidized (heme iron 3+) soybean leghemoglobin(FIG. 4 panel A) and equine heart muscle myoglobin (FIG. 4 panel B)showing similarity of UV-VIS absorption profiles of two proteins. Wepurified soybean leghemoglobin from soybean root-nodules using heredescribed protocol. Purified equine myoglobin was purchased fromSigmaAldrich. Soybean leghemoglobin (FIG. 4 panel A) and equinemyoglobin (FIG. 4 panel B) were reduced with 1 mm sodium hydrosulfite.Shown are UV-VIS absorption spectra of heme Fe3+(blue line) and hemeFe2+(red line) of soybean leghemoglobin (FIG. 4 panel A) and equinemyoglobin (FIG. 4 panel B). Insets show a zoom-in of UV-VIS spectra in450 nm to 700 nm region. (FIG. 4 panel C) Images of 10 ul liquid dropletof a 40 mg/ml solution of soybean leghemoglobin in the heme-Fe3+ state(left droplet) showing characteristic rusty red color and a 40 mg/mlsolution of soybean leghemoglobin solution in the heme-Fe2+ state (rightdroplet) showing characteristic red color of and (right image)corresponding samples of equine myoglobin.

In other embodiments, leghemoglobin can be sourced from non-plantsources, such as from organisms such as bacteria or yeast which havebeen genetically modified to express high levels of leghemoglobin.

The oxidation state of the iron ion in leghemoglobin is important forits color. Leghemoglobin with the heme iron in the +2 oxidation stateappears vivid red in color, while leghemoglobin with the heme iron inthe +3 oxidation state appears brownish red. Thus, in usingleghemoglobin as a source of red color in a meat replica, it isdesireable to reduce the heme iron from the +3 state to the +2 state.Heme iron in leghemoglobin can be switched from oxidized (+3) state toreduced (+2) state with reducing reagents. Examples of successfulreduction of leghemoglobin heme iron with sodium hydrosulfite andtitanium citrate are illustrated in FIG. 5. In FIG. 5 the UV-VISspectrogram of purified soybean leghemoglobin in which the heme iron isin the oxidized (+3) state is represented by the blue curves in eachpanel. The red curves in each panel represent the UV-VIS spectra of thesame leghemoglobin species after reduction to the (+2) state (red lines)by addition of (Panel A) 1 mM sodium hydrosulfite or (Panel B) 0.24%(wt/v) titanium citrate in 20 mM potassium phosphate pH 7.3, 100 mMsodium chloride buffer. The Insets show a zoom-in of UV-VIS spectra in450-700 Nm region. For this example, leghemoglobin was purified fromsoybean root nodules using 60/90% ammonium sulfate fractionation andexchanged into 20 mM potassium phosphate pH 7.4, 100 mM sodium chloridebuffer. Sodium hydrosulfite stock solution was prepared by dissolving100 mM sodium hydrosulfite in 1 mM sodium hydroxide in water. Titaniumcitrate stock solution was prepared from 20% (wt/v) Ti-chloride inhydrochloric acid by mixing it with 0.2M sodium citrate (1:10 v/v). pHwas adjusted using sodium carbonate to pH 7.0.

Leghemoglobin can be purified from legume root nodules, such as the rootnodules of peas or soybeans (FIG. 1 shows Leghemoglobin isolated frompea root nodules). Root nodules from soy beans were thoroughly cleanedto remove soil and extraneous root tissues prior to root nodule lysis in20 mM potassium phosphate pH 7.4, 100 mM sodium chloride, 1 mm EDTA and1 mM ascorbic acid. Root nodules were lysed by grinding root-nodulesusing a Vitamix blender. For some samples Polyvinylpyrrolidone polymerwas added at 30% wt/v to aid in removal of plant phenolic smallmolecules that mediate oxidation of leghemoglobin heme-iron. Root nodulelysate was fractionated using ammonium sulfate in two steps, firstammonium sulfate was added to 60% wt/v. Pellet was discarded andsupernatant brought to 90% wt/v. ammonium sulfate. Leghemoglobin wascollected as a precipitated pellet in 90% ammonium sulfate fractionAmmonium sulfate precipitated leghemoglobin was resuspended in 20 mMpotassium phosphate, 1 mM EDTA, 50 mM sodium chloride and ammoniumsulfate was removed using dialysis or size-exclusion chromatography inthe same buffer. In some instances this was the last purification step,while in other instances leghemoglobin was further purified usinganion-exchange chromatography (FFQ GE Healthcare), which was sometimesfollowed by size-exclusion chromatography (Sephacryl S-100, GEHealthcare). Soybean leghemoglobin from 90% ammonium sulfate pellet wasloaded on anion exchange columns (FFQ or DEAE, GE Healthcare) indifferent buffers (20 mM potassium phosphate pH 7.4, containing 0 to 100mM sodium chloride, 20 mM Tris pH 8 containing 0 to 100 mM sodiumchloride, 20 mM sodium borax pH 9.8, 20 mM sodium chloride, 20 mM sodiumcarbonate pH 9, 20 mM sodium chloride) and purified either inflow-through or using sodium chloride (0-1M salt gradient). An exampleof the leghemoglobin purification flow from soybean root nodules isrepresented in FIG. 6. The figure shows SDS-PAGE fractionation ofdifferent soybean leghemoglobin purification steps (Lane 1) Soybeanroot-nodule lysate; (Lane 2) Soybean root-nodule lysate purified by60/90% (wt/v) ammonium sulfate fractionation. Shown is the proteincontent of 90% ammonium sulfate fractionated protein pellet resuspendedin 20 mm potassium phosphate pH 7.4, 100 mM sodium chloride, 1 mM EDTAbuffer; Proteins from 90% ammonium sulfate pellet were further purifiedby anion-exchange chromatography (FFQ GE Healthcare) in 20 mM potassiumphosphate ph 7.4, 100 mM sodium chloride. Leghemoglobin collected in theflowthrough is shown in Lane 3. Anion-exchange flowthrough wasfractionated using size-exclusion chromatography (Sephacryl 5-100 GEHealthcare) and resulting leghemoglobin fraction is shown in Lane 4.Leghemoglobin content at different purification steps was determined bydetermining the fraction of leghemoglobin band on SDS-PAGE gel in arespective sample using ImageDoc analysis software (BioRad). Purity(partial abundance) of leghemoglobin at respective steps in thepurification steps was: lysate: 32.7% (lane 1), 60/90% (wt/v) ammoniumsulfate fractionation 78% (lane 2), anion-exchange chromatography ˜83%(lane 3), and size-exclusion chromatography to ˜95% (lane 4).

Leghemoglobin can also be produced by genetically engineering abacterium or fungus to produce it. One illustrative example is shown inFIG. 7. FIG. 7 shows stained SDS-PAGE gel analysis of (A) soybeanleghemoglobin expressed and purified using recombinant proteintechnology and (B) soybean leghemoglobin purified from soybean rootnodules. (A) Recombinant Soybean leghemoglobin A carrying His-tag andTEV protease His-tag removal site was expressed in E. coli BL21 strainand purified using His-tag affinity chromatography (Talon resin,CloneTech). The left lane contains molecular weight standards, the rightlane contains purified recombinant soybean leghemoglobin A (arrow).Expected molecular weight of the recombinant soybean leghemoglobin A is17.1 kDa. (B) SDS-PAGE gel of purified Soybean leghemoglobin from rootnodules. The left lane contains molecular weight standards, the rightlane contains purified soybean leghemoglobin A (arrow). Massspectrometry analysis of purified material determined that all foursoybean leghemoglobin isoforms are present, and are full length (datanot shown). Expected molecular weights (MW) of soybean leghemoglobinisoforms range from MW15.4 to 15.8 kDa.

Leghemoglobin purified from soybean and fava root nodules, respectivelywas tasted by a panel of volunteers and in each case described astasting like blood.

Leghemoglobin can be isolated from the roots nodules of legumes such assoy beans, fava beans, cow peas, lima beans, garbanzo beans, peas,lupine, lotus japonicum or other legumes. The root nodule (for exampleroot nodules from a pea plant) is obtained and homogenized in an aqueoussolution, soluble proteins including leghemoglobin are recovered afterinsoluble matter is removed by precipitation or filtration.Leghemoglobin can be purified by selective precipitation and/orchromatography and/or the use of molecules with specific affinity forleghemoglobin. (FIG. 1, showing 100 mls of solution of leghemoglobinisolated from 30 grams of pea root nodules).

Heme proteins, for example leghemoglobin, can be combined with otherplant based meat replica components. In some embodiments the hemeproteins are captured in a gel which contains other components, forexample lipids and or proteins. In some aspects a multiple gels arecombined with non-gel based heme proteins. In some embodiments thecombination of the heme proteins and the other compounds of theconsumable are done to insure that the heme proteins are able to diffusethrough the consumable. In some embodiments the consumable is ed in aheme-protein containing solution, for instance a leghemoglobin solution.In some embodiments the consumable is soaked in a heme proteincontaining solution, for instance a leghemoglobin solution for 1, 5, 10,15, 20 or 30 hours. In some embodiments the consumable is soaked in aheme containing solution, for instance a leghemoglobin solution for 1,5, 10, 15, 30, or 45 minutes.

FIG. 8 shows an example of 6 cubes of a commercial meat analog (Quornchicken analog), about 1 cm on a side, 4 of which (Left and lower right)have been soaked in a solution of about 10 mg/ml soybean leghemoglobinin 20 mM Potassium phosphate pH 7.4 and 100 mM NaCl; the remaining two(Upper right) were soaked in the same buffer without leghemoglobin. Notethe deep pink color of the leghemoglobin-infused cubes in contrast tothe pale tan color of the un-infused cubes.

FIG. 9 shows the 4 leghemoglobin-infused cubes of Quorn chicken analogin the process of cooking in a pan at 350° C. The two lower cubes havebeen turned over to expose the grilled surface, which has turned brown.Note in the upper two cubes that the heated portion has turnedgrey-brown, while the cooler top surface retains its pink color. In someembodiments the consumable is injected with a heme containing solution,for instance a leghemoglobin solution, until the consumable is the colorof uncooked meat.

Given the usefulness of heme proteins for coloring consumables it willbe useful to detect whether a product contains a particular hemeprotein. Accordingly the present invention includes in some embodimentsmethods to determine whether a product contains a heme protein. Methodsfor detecting proteins are well known in the art. For example an ELISAor proximity-ligation assoacy or luninex assay or western blot analysiscan be performed to determine whether leghemoglobin is present in a foodproduct such as meat or a meat replica. In one embodiment the detectionmethods are performed to determine whether meat has been altered withleghemoglobin.

Examples

An exemplary muscle replica composition comprising one or more isolated,purified plant proteins is described herein.

Protein Purification for Components of the Replica

Moong bean seeds, Green Pea dry seed were purchased as milled flour andused for purification of respective seed storage proteins. Rubsico waspurified from fresh alfalfa plant. Protein composition at individualfractionation steps was monitored by SDS-PAGE and protein concentrationswere measured by standard UV-VIS and Pierce assay methods.

Moong bean 8S globulins: Moong bean flour was resuspended in 50 mMpotassium phosphate buffer pH 7 and 0.5M NaCl at 1:4 (wt/v) ratio, andmixture was incubated for 1 hr. Unsoluble material was separated bycentrifugation and proteins in the supernatant were fractionated byaddition of ammonium sulfate in 2 steps: 50% (wt/v) followed by 90%(wt/v). Protein precipitated in 90% fraction contained the moong bean 8Sglobulins and was stored at −20 C until further use.

Pea-albumins: Green pea dry seed flour was resuspended at 1:10 (wt/v)ratio in 50 mM sodium acetate buffer pH 5 and incubated for 1 hr.Unsoluble material was separated by centrifugation and proteins in thesupernatant were fractionated by ammonium sulfate precipitation in twosteps: 50% (wt/v) followed by 90% (wt/v) Ammonium sulfate solutions werestirred for 1 hour and ammonium sulfate precipitated proteins removed bycentrifugation. Proteins of interest precipitated in 90% (wt/v) ammoniumsulfate. Pellet was stored at −20 C until further use.

Pea-globulins: Green pea dry seed flour was resuspended at 1:10 (wt/v)ratio in 20 mM potassium phosphate buffer pH 8, 0.4M sodium chloride andstirred for 1 hr. After centrifugation, the supernatant was subjected toammonium sulfate fractionation. First, supernatant was brought to 50%(wt/v) ammonium sulfate, and precipitated proteins removed. Second, 50%(wt/v) ammonium sulfate supernatant was brought to 80% (wt/v) ammoniumsulfate saturation. The 80% (wt/v) ammonium sulfate pelleted proteincontained globulins of interest. Pellet was stored at −20° C. untilfurther use.

RuBisCO: RuBisCO was fractionated from alfalfa greens (or other greenplants eg soybean plants, spinach etc) by first grinding leaves with 4volumes of cold 50 mM KPhosphate buffer pH 7.4 buffer (with (in lab) orwithout (in field) 0.5M NaCl+2 mM DTT+1 mM EDTA) in a blender. Theresulting slurry was centrifuged to remove debris, and the supernatant(crude lysate) was used in further purification steps. Proteins in thecrude lysate were fractionated by addition of ammonium sulfate to 30%(wt/v) saturation. The solution was stirred for 1 hr and thencentrifuged. The pellet from this step was discarded and additionalammonium sulfate was added to the supernatant to 50% (wt/v) ammoniumsulfate saturation. The solution was centrifuged again after stirringfor 1 hr. The pellet from this step contains RuBisCO, and was kept at−20 C until used.

Obtaining Plant Proteins.

Moong bean seed 8S protein was purified by ammonium sulfatefractionation as described. Pellet was resuspended in 20 mM potassiumphosphate pH 7.4 and 0.5M sodium chloride and ammonium sulfate removedby dialysis against the same buffer. Any precipitate was removed bycentrifugation at 16 000 g, 10 min and protein concentrated to desiredconcentration. Pea globulins purified by ammonium sulfate fractionationas described. Protein pellet was resuspended in 20 mM potassiumphosphate pH 7.4 and 0.4M sodium chloride and ammonium sulfate removedby dialysis against the same buffer. Any precipitate was removed bycentrifugation at 16 000 g, 10 min and protein concentrated to desiredconcentration. Pea albumin purified by ammonium sulfate fractionation asdescribed. Protein pellet was resuspended in 20 mM potassium phosphatepH 7.4 and 0.1M sodium chloride and ammonium sulfate removed by dialysisagainst the same buffer. Any precipitate was removed by centrifugationat 16 000 g, 10 min and protein concentrated to desired concentration.

Constructing a Muscle Tissue Analog

Moong bean seed 8S protein was purified by ammonium sulfatefractionation as described above, For preparation of gels, 200 g ofpellet was dissolved in 400 ml of dialysis buffer (20 mM potassiumphosphate, 400 mM NaCl, pH 7.3) and the resulting solution dialyzed for6 hours against 5 l of dialysis buffer, replaced twice with freshbuffer. Protein solution was centrifuged at 12,000 g for 15 min toremove debris. Protein was concentrated by dialyzing for 36 hoursagainst 5 l of 30% w/w solution of PEG 8000 (polyethylene glycol,molecular weight 8000) in dialysis buffer. Final protein concentrationwas 150 mg/ml.

Leghemoglobin was purified from soybean root nodules. Legume rootnodules were cleaned to remove soil and extraneous root tissues prior toroot nodule lysis in 20 mM potassium phosphate pH 7.4, 100 mM sodiumchloride, 1 mm EDTA and 1 mM ascorbic acid. Root nodules were lysed bygrinding root-nodules using juicer blender. Unsoluble material wasseparated by centrifugation. Root nodule lysate was fractionated usingammonium sulfate in two steps, first ammonium sulfate was added to 60%wt/v and solution incubated for 1 hr, 4′C. Pellet was discarded andsupernatant brought to 90% wt/v ammonium sulfate and incubated for 12hr, 4′C. Leghemoglobin was collected as a precipitated pellet in 90%ammonium sulfate fraction and resuspended in 20 mM potassium phosphate,1 mM EDTA, 100 mM sodium chloride. SDS-PAGE gel analysis determined thatprotein solution contains 70% leghemoglobin and 30% other root noduleproteins Ammonium sulfate was removed using size-exclusionchromatography in the same buffer. Leghemoglobin was concentrated bydialyzing for 48 hr against 30% PEG 8000 (polyethylene glycol, molecularweight 8000) in 20 mM potassium phosphate pH 7.3, 100 mM sodiumchloride. Total protein concentration was 57 mg/ml. UV-VIS spectrasuggested that leghemoglobin was in heme-iron oxidized state. Thus,leghemoglobin was incubated with 5 mM sodium hydrosulfite for 5 min andsodium hydrosulfite was removed using size-exclusion chromatography in20 mM potassium phosphate, 100 mM sodium chloride buffer. Leghemoglobinwas further concentrated to 35.4 mg/ml. UV-VIS spectra analysisconfirmed that leghemoglobin is in heme-iron reduced state.

Transglutaminase was obtained commercially from (Activa TI, Ajimoto).Stock solution (20% wt/v) was made in 20 mM potassium phosphate pH 7.3,100 mM sodium chloride buffer.

To prepare “dark” muscle tissue analog (FIG. 10), 43 ml of moong beanprotein solution (150 mg/ml in dialysis buffer) were mixed with 37 ml ofleghemoglobin solution (46.5 mg/ml leghemoglobin and 20 mg/ml of othersoybean root nodule protein) in 20 mM potassium phosphate, 100 mM NaCl,pH 7.3). 20 ml of transglutaminase solution (20% w/w) were added,solutions thoroughly mixed, divided into two 50 ml Falcon tubes andincubated overnight at room temperature. Final protein concentrationswere 65 mg/ml for moong bean protein, 18 mg/ml of leghemoglobin, 91mg/ml total protein.

“White” muscle analog (FIG. 11) was prepared by mixing 43 ml moong beanprotein solution (150 mg/ml) with 45 ml of 11.7 mg/ml solution ofleghemoglobin and 0.8% (wt/v) of transglutaminase solution. Finalprotein concentrations were 63 mg/ml for moong bean protein, 5.2 mg/mlof leghemoglobin, 68 mg/ml total protein.

The “dark” muscle tissue analog formed an opaque gel of dark chocolatecolor, smooth uniform texture, with glistening surface, and a smallamount (<1 ml) of dark red, venous blood colored liquid on top. The gelwas freely standing, elastic but fragile, similar in appearance to thinJell-O. The gel has a medium aroma with notes of beans and blood clearlydiscernible. The flavor is dominated by notes of beans and iron/blood,with weaker grassy and medicinal/chemical flavors. The taste is salty,with a long aftertaste of blood.

The “white” muscle tissue analog was very similar, but with muchlighter, cappuccino-like, color. It was also more fragile, 2-3-fold lessstrong against compression.

Fat Tissue Analog

Fat tissue analog using moong bean 8S globulin fraction was prepared asfollows: 15 ml of moong bean protein solution (150 mg/ml in dialysisbuffer) were mixed with 15 ml of rice bran oil. 6 ml of transglutaminasesolution (20% w/w) were added, solutions thoroughly emulsified using ahomogenizer (VWR) at speed #2. Emulsion was aliquoted into 1.6 eppendorftubes and incubated overnight at room temperature. After that, tubeswere heated at 95° C. for 5 min in a heat block, and allowed to cooldown to room temperature on a bench. Final concentrations were 75 mg/mlfor moong bean protein, 50% w/w oil.

Fat tissue analog using pea globulin (100 mg/ml) was prepared by thesame method.

Additionally, fat tissue analog was prepared from pea globulin, andeither rice bran or canola oil, in bulk by the same method, but withoutaliquoting emulsions into eppendorf tubes. Instead, emulsions in 50 mlFalcon tubes were rotated overnight on a nutator, and were subsequentlyincubated at 90° C. for 30 min.

Fat tissue analog based on moong beans (FIG. 12) and prepared ineppendorf tubes formed an opaque gel of off-white color, smooth uniformtexture, with no visible discernible liquid that was not incorporatedinto the gel. The gel was freely standing, elastic and springy. The gelhas a slight, pleasant aroma and a mild and pleasant flavor. The tasteis mildly salty.

Fat tissue analog based on pea globulin (FIG. 13) and prepared ineppendorf tubes was very similar to moong bean-based fat analog, exceptthat it gave up a little of oil upon compression. Fat tissue analogprepared in 50 ml Falcon tubes were similar in appearance, texture andaromas, but substantially softer (2-fold softer for canola oil, and3-fold softer for rice bran oil, according to compressibilitymeasurements).

Connective Tissue Analog

Connective tissue analog prototypes were developed using zein proteinsourced from 100% yellow corn gluten meal, or from commercial sources,such as Amazein (Prairie Gold, Bloomington, Ill.). Zein proteins weresolubilized in 70-90% ethanol with desired ratios at 1:3 to 1:5(solids:solution). By precipitating zein proteins, for example by achange in pH, in a controlled manner, large zein structures result withphysicochemical properties that can be manipulated as desired. Forexample, FIG. 14 shows connective-tissue analog strands that werecreated using a 1:3 ratio in 70% ethanol, loaded into a syringe with a23 gauge needle (ID 0.337 mm) The solution was slowly extruded from thebottom of a 5 inch-high vessel into an excess of 5 M NaCl solution. Theethanol-zein solution being less dense than the NaCl solution, floatedupward, drawing out a fibrous stand of solidifying zein. The NaCl wasconstantly stirred as the strands began to develop to assist in thestrand lengthening. The strands bunch together and become a hard, densemass.

Ground beef replica prototypes made from gels of plant proteins andplant oils.

A ground beef prototype patty was made by combining 62% (wt/wt) muscleanalog (62% (wt/wt) “dark muscle analog” and 38% (wt/wt) “white muscleanalog”), 29% (wt/wt) fat tissue analog (from pea globulin and canolaoil), 5% (wt/wt) connective tissue analog (FIG. 15 panel A). A groundbeef prototype patty was made by combining 62% muscle analog (62% “darkmuscle analog” and 38% “white muscle analog), 29% fat tissue analog(from moong bean seed 8S protein and rice bran oil), 5% connectivetissue analog (FIG. 15 panel B). A ground beef prototype patty was madeby combining 71% (wt/wt) muscle tissue analog (composed of 60% “white”muscle analog, 40% “dark” muscle analog), 23% fat tissue (from pea seedglobulin proteins and canola oil) (FIG. 15 panel C). A ground beefprototype patty was made by combining 67% “White” muscle analog, with28% fat tissue analog (from pea globulins and rice bran oil), (FIG. 15,panel D)

In a further test, the effect of cooking the ground beef replica pattieswas evaluated by grilling on a 350° F. pan. A ground beef patty analogwas made by combining 62% (wt/wt) muscle tissue analog (62% (wt/wt)“dark muscle analog” and 38% (wt/wt) “muscle analog”), 29% (wt/wt) fattissue analog (from pea globulin and canola oil), 5% (wt/wt) connectivetissue analog (FIG. 16). The panel on the left shows the patty beforecooking and the panel on the right shows the same patty after cookingfor about 2 minutes. Observers described the aroma of the cooking groundbeef replica as distinctly “beefy”.

1. (canceled)
 2. A consumable food product comprising an isolatediron-containing protein and an isolated plant protein.
 3. The consumablefood product of claim 2, wherein the iron-containing protein is a hemeprotein.
 4. The consumable food product of claim 2, wherein theiron-containing protein is selected from the group consisting of ahemoglobin, a myoglobin, a leghemoglobin, a non-symbiotic hemoglobin, achlorocruorin, an erythrocruorin, a neuroglobin, a cytoglobin, aprotoglobin, a truncated 2/2 globin, HbN, a cyanoglobin, HbO, Glb3, acytochrome, a Hell's gate globin I, a bacterial hemoglobin, a ciliatemyoglobin, and a flavohemoglobin.
 5. The consumable food product ofclaim 2, wherein the iron-containing protein comprises an amino acidsequence with at least 70% homology to SEQ ID NO
 1. 6. The consumablefood product of claim 2, wherein the iron-containing protein has aUV-VIS spectrum that is substantially similar to that of myoglobin froman animal.
 7. The consumable food product of claim 2, wherein theisolated plant protein is selected from the group consisting of aribosomal protein, a hexokinase, a lactate dehydrogenase, a fructosebisphosphate aldolase, a phosphofructokinases, a triose phosphateisomerase, a phosphoglycerate kinase, a phosphoglycerate mutase, anenolase, a pyruvate kinase, a glyceraldehyde-3-phosphate dehydrogenase,a pyruvate decarboxylases, an actin, a translation elongation factor,ribulose-1,5-bisphosphate carboxylase oxygenase,ribulose-1,5-bisphosphate carboxylase oxygenase activase (rubiscoactivase), a seed storage protein, an oleosin, a caloleosin, asteroleosin, vegetative storage protein A, and vegetative storageprotein B.
 8. The consumable food product of claim 7, wherein the seedstorage protein is selected from an albumin, a glycinin, a conglycinin,a globulin, a vicilin, a conalbumin, a gliadin, a glutelin, a gluten, aglutenin, a hordein, a prolamin, a phaseolin, a proteinoplast, asecalin, a triticeae gluten, and a zein, and a mixture of two or morethereof.
 9. The consumable food product of claim 8, wherein, when theseed storage protein is conglycinin, the consumable food productcontains between 2% and 6% by weight of the seed storage protein. 10.The consumable food product of claim 2, further comprising a fat. 11.The consumable food product of claim 10, wherein the fat is selectedfrom the group consisting of corn oil, olive oil, soy oil, peanut oil,walnut oil, almond oil, sesame oil, cottonseed oil, rapeseed oil, canolaoil, safflower oil, sunflower oil, flax seed oil, algal oil, palm oil,palm kernel oil, coconut oil, babassu oil, shea butter, mango butter,cocoa butter, wheat germ oil, rice bran oil, an oil produced bybacteria, an oil produced by archaea, an oil produced by fungi, an oilproduced by genetically engineered bacteria, an oil produced bygenetically engineered algae, an oil produced by genetically engineeredarchaea, and an oil produced by genetically engineered fungi, and amixture of two or more thereof.
 12. The consumable food product of claim2, wherein the consumable food product is characterized by one or moreof the following: contains no methylcellulose, contains no carrageenan,contains no caramel color, contains no konjac flour, contains no gumarabic, contains no acacia gum, contains less than 1% wheat gluten,contains no soy protein isolate, contains no soy protein concentrate,contains no soy protein, contains less than 5% carbohydrates, containsno tofu, contains less than 1% cellulose, contains no cholesterol, orcontains less than 5% insoluble carbohydrates.
 13. The consumable foodproduct of claim 2, wherein the consumable food product contains between5% and 30% by weight of the isolated plant protein.
 14. The consumablefood product of claim 2, wherein the consumable food product containsabout 0.05% to about 0.6% by weight of the isolated iron-containingprotein.
 15. The consumable food product of claim 2, wherein theisolated iron-containing protein is leghemoglobin.
 16. The consumablefood product of claim 2, wherein the consumable food product comprises aplant-based meat replica.
 17. The consumable food product of claim 2,wherein the consumable food product comprises an animal meat product.18. A meat substitute product comprising an isolated iron-containingprotein.
 19. The meat substitute product of claim 18, wherein the meatsubstitute product comprises an animal meat product.
 20. The meatsubstitute product of claim 18, wherein the meat substitute product,before it is cooked, accurately mimics the color of an equivalent meatproduct in its raw state, and after it is cooked, accurately mimics thecolor of the equivalent meat product in the cooked state.
 21. The meatsubstitute product of claim 18, wherein the meat substitute product,after it is cooked, further accurately mimics one or more of thetexture, aroma, and taste of the equivalent meat product in the cookedstate.
 22. The meat substitute product of claim 18, wherein theiron-containing protein is selected from the group consisting of ahemoglobin, a myoglobin, a leghemoglobin, a non-symbiotic hemoglobin, achlorocruorin, an erythrocruorin, a neuroglobin, a cytoglobin, aprotoglobin, a truncated 2/2 globin, HbN, a cyanoglobin, HbO, Glb3, acytochrome, a Hell's gate globin I, a bacterial hemoglobin, a ciliatemyoglobin, and a flavohemoglobin.
 23. The meat substitute product ofclaim 18, wherein the isolated iron-containing protein is leghemoglobin.24. The meat substitute product of claim 18, wherein the meat substituteproduct contains about 0.05% to about 0.6% by weight of the isolatediron-containing protein.
 25. The meat substitute product of claim 18,further comprising an isolated plant protein.
 26. The meat substituteproduct of claim 25, wherein the isolated plant protein is selected fromthe group consisting of a ribosomal protein, a hexokinase, a lactatedehydrogenase, a fructose bisphosphate aldolase, a phosphofructokinases,a triose phosphate isomerase, a phosphoglycerate kinase, aphosphoglycerate mutase, an enolase, a pyruvate kinase, aglyceraldehyde-3-phosphate dehydrogenase, a pyruvate decarboxylases, anactin, a translation elongation factor, ribulose-1,5-bisphosphatecarboxylase oxygenase, ribulose-1,5-bisphosphate carboxylase oxygenaseactivase (rubisco activase), a seed storage protein, an oleosin, acaloleosin, a steroleosin, vegetative storage protein A, and vegetativestorage protein B.
 27. The meat substitute product of claim 26, whereinthe seed storage protein is selected from an albumin, a glycinin, aconglycinin, a globulin, a vicilin, a conalbumin, a gliadin, a glutelin,a gluten, a glutenin, a hordein, a prolamin, a phaseolin, aproteinoplast, a secalin, a triticeae gluten, and a zein, and a mixtureof two or more thereof.
 28. The meat substitute product of claim 27,wherein, when the seed storage protein is conglycinin, the consumablefood product contains between 2% and 6% by weight of the seed storageprotein.
 29. The meat substitute product of claim 20, wherein theequivalent meat product is beef.
 30. The meat substitute product ofclaim 29, wherein the equivalent meat product beef is in the form ofground beef.